Replication competent hepatitis C virus and methods of use

ABSTRACT

The present invention provides a replication competent hepatitis C virus that includes a heterologous polynucleotide. The invention also includes methods for modifying a hepatitis C virus polynucleotide, selecting a replication competent hepatitis C virus polynucleotide, detecting a replication competent hepatitis C virus polynucleotide, and identifying a compound that inhibits replication of a hepatitis C virus polynucleotide.

CONTINUING APPLICATION DATA

This application claims priority to U.S. patent application Ser. No.09/747,419, filed Dec. 23, 2000, which claims the benefit of U.S.Provisional Application Ser. No. 60/171,909, filed Dec. 23, 1999, eachof which are incorporated by reference herein. This application alsoclaims the benefit of U.S. Provisional Application Ser. No. 60/325,236,filed Sep. 27, 2001, and Ser. No. 60/338,123, filed Nov. 13, 2001, eachof which are incorporated by reference herein.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.U19-A140035, awarded by the National Institute of Allergy and InfectiousDiseases. The Government has certain rights in this invention.

BACKGROUND

Hepatitis C virus is the most common cause of chronic viral hepatitiswithin the United States, infecting approximately 4 million Americansand responsible for the deaths of 8,000-10,000 persons annually due toprogressive hepatic fibrosis leading to cirrhosis and/or the developmentof hepatocellular carcinoma. Hepatitis C virus is a single stranded,positive-sense RNA virus with a genome length of approximately 9.6 kb.It is currently classified within a separate genus of the flavivirusfamily, the genus Hepacivirus. The epatitis C virus genome contains asingle large open reading frame (ORF) that follows a 5′ non-translatedRNA of approximately 342 bases containing an internal ribosome entrysegment (IRES) directing cap-independent initiation of viraltranslation. The large ORF encodes a polyprotein which undergoespost-translational cleavage, under control of cellular and viralproteinases. This yields a series of structural proteins which include acore or nucleocapsid protein, two envelope glycoproteins, E1 and E2, andat least six nonstructural replicative proteins. These include NS2(which with the adjacent NS3 sequence demonstrates cis-activemetalloproteinase activity at the NS2/NS3 cleavage site), NS3 (a serineproteinase/NTPase/RNA helicase), NS4A (serine proteinase accessoryfactor), NS4B, NS5A, and NS5B (RNA-dependent RNA polymerase).

With the exception of the 5′ non-translated RNA, there is substantialgenetic heterogeneity among different stains of hepatitis C virus.Phylogenetic analyses have led to the classification of epatitis C virusstrains into a series of genetically distinct “genotypes,” each of whichcontains a group of genetically related viruses. The genetic distancebetween some of these genotypes is large enough to suggest that theremay be biologically significant serotypic differences as well. There islittle understanding of the extent to which infection with a virus ofany one genotype might confer protection against viruses of a differentgenotype.

Several types of human interferon have proven effective in the treatmentof infection by hepatitis C virus, either alone as monotherapy, or incombination with ribavirin. However, treatment with interferon-ribavirincarries a high risk of treatment failure, either primary failure ofvirus elimination, or relapse of the infection upon cessation oftherapy. Moreover, these therapeutic agents are relatively toxic and areassociated with a high frequency of adverse reactions. The developmentof better (more effective and safer) antiviral agents capable ofsuppressing or eliminating hepatitis C virus infection has been hinderedby the fact that this virus replicates with very low efficiency, or notat all, in cultured cells. The absence of a highly permissive cellculture system that is capable of supporting robust replication of thevirus has prevented the development of high throughput antiviral screensfor use in the development of inhibitors of viral replication, and hasdelayed the investigation of the virus and relevant aspects of itsmolecular and cellular biology. It has also stymied efforts at vaccinedevelopment and the immunologic characterization of the virus, the humanresponse to hepatitis C virus, and the diseases associated withinfection. The development of infectious molecular cDNA clones of theviral genome has done little to solve this problem, since virus can berescued from the RNA transcribed from such clones only by its injectioninto the liver of a living chimpanzee or other susceptible primate.

SUMMARY OF THE INVENTION

The present invention provides methods for identifying a compound thatinhibits replication of an HCV RNA. The methods include contacting acell that contains a replication competent HCV RNA with a compound. Thereplication competent HCV RNA includes a heterologous polynucleotidethat contains a first coding sequence encoding a transactivator. Thetransactivator may inlcude an amino acid sequence having at least about70% identity with the amino acid sequence SEQ ID NO:19 or amino acids4-89 of SEQ ID NO:21. The cells are incubated under conditions where thereplication competent HCV RNA replicates in the absence of the compound,and the replication competent HCV RNA is detected. A decrease thereplication competent HCV RNA in the cell contacted with the compoundcompared to the replication competent HCV RNA in a cell not contactedwith the compound indicates the compound inhibits replication of thereplication competent HCV RNA.

The HCV RNA may include a second coding sequence encoding a hepatitis Cvirus polyprotein and a 3′ non-translated RNA, and the heterologouspolynucleotide may be present in the 3′ non-translated RNA or 5′ of thesecond coding sequence. Alternatively, the HCV RNA may include a 3′non-translated RNA and a second coding sequence encoding a subgenomichepatitis C virus polyprotein, and the heterologous polynucleotide maybe present in the 3′ non-translated RNA or 5′ of the second codingsequence.

The heterologous polynucleotide may include a second coding sequenceencoding a selectable marker, and the first coding sequence and thesecond coding sequence together encode a fusion polypeptide. Theheterologous polynucleotide may further include a third coding sequenceencoding a cis-active proteinase present between the first codingsequence encoding the transactivator and the second coding sequenceencoding the selectable marker. The first coding sequence, the thirdcoding sequence, and the second coding sequence together encode a fusionpolypeptide.

The cell may include a polynucleotide that includes a transactivatedcoding sequence encoding a detectable marker and an operator sequenceoperably linked to the transactivated coding sequence. Thetransactivator interacts with the operator sequence and altersexpression of the transactivated coding sequence. Detecting thereplication competent HCV RNA in the cell includes detecting thedetectable marker encoded by the transactivated coding sequence. Thepresent invention is also directed to the cell.

The present invention also provides a method for selecting a replicationcompetent HCV RNA. The method includes incubating a vertebrate cell inthe presence of a selecting agent, for instance, an antibiotic. The cellincludes an HCV RNA that includes a first coding sequence encoding ahepatitis C virus polyprotein, and a heterologous polynucleotide, andthe heterologous polynucleotide includes a second coding sequenceencoding a selectable marker that confers resistance to the selectingagent. The selecting agent inhibits replication of a cell that does notexpress the selectable marker. A cell that replicates in the presence ofthe selecting agent is detected, and the presence of such a cellindicates the HCV RNA is replication competent.

The method may further include obtaining a virus particle produced bythe first cell and exposing a second vertebrate cell to the isolatedvirus particle and incubating the second vertebrate cell in the presenceof the selecting agent. A second cell that replicates in the presence ofthe selecting agent is detected, wherein the presence of such a cellindicates the HCV RNA present in the first cell produces an infectiousvirus particle.

The HCV RNA may include a 3′ non-translated RNA, and the heterologouspolynucleotide may be present in the 3′ non-translated RNA or 5′ of thefirst coding sequence.

The present invention also provides a method for detecting a replicationcompetent HCV RNA. The method includes incubating a vertebrate cellcomprising an HCV RNA. The HCV RNA includes a first coding sequenceencoding a hepatitis C virus polyprotein, or a subgenomic hepatitis Cvirus polyprotein, and a heterologous polynucleotide includes a secondcoding sequence encoding a transactivator. The cell includes atransactivated coding region and an operator sequence operably linked tothe transactivated coding region, where the transactivated coding regionencodes a detectable marker and the transactivator alters transcriptionof the transactivated coding region. The detectable marker is detected,and the presence of the detectable marker indicates the cell contains areplication competent HCV RNA.

The heterologous polynucleotide may further include a third codingsequence encoding a selectable marker, and the second coding sequenceand the third coding sequence together encode a fusion polypeptide.Alternatively, the heterologous polynucleotide may further include afourth coding sequence encoding a cis-active proteinase present betweenthe second coding sequence encoding the transactivator and the thirdcoding sequence encoding the selectable marker, and the second codingsequence, the fourth coding sequence, and the third coding sequencetogether encode a fusion polypeptide.

The present invention further provides replication competent HCVpolynucleotides that include a first coding sequence encoding asubgenomic hepatitis C virus polyprotein, and a heterologouspolynucleotide containing a second coding sequence encoding atransactivator, wherein the heterologous polynucleotide is located 5′ ofthe first coding sequence. In another aspect, the present inventionprovides a replication competent HCV polynucleotide containing a firstcoding sequence encoding a hepatitis C virus polyprotein, and aheterologous polynucleotide.

The present invention also provides kits. The kits include a replicationcompetent HCV polynucleotide containing a heterologous polynucleotidethat has a first coding sequence encoding a transactivator, and avertebrate cell that includes a polynucleotide containing atransactivated coding sequence encoding a detectable marker and anoperator sequence operably linked to the transactivated coding sequence.The transactivator interacts with the operator sequence and altersexpression of the transactivated coding sequence.

Definitions

As used herein, the term “HCV” refers to a hepatitis C virus, e.g., aviral particle, or a polynucleotide that includes a hepatitis C viralgenome or a portion thereof. Preferably, the polynucleotide is RNA.

As used herein, the term “replication competent” refers to an HCV RNAthat replicates, e.g., HCV nucleic acid is synthesized, for instancesynthesis of the negative-sense strand, in vitro or in vivo. As usedherein, the term “replicates in vitro” indicates the HCV RNA replicatesin a cell that is growing in culture. The cultured cell can be one thathas been selected to grow in culture, including, for instance, animmortalized or a transformed cell. Alternatively, the cultured cell canbe one that has been explanted from an animal. “Replicates in vivo”indicates the HCV RNA replicates in a cell within the body of an animal,for instance a primate (including a chimpanzee) or a human. In someaspects of the present invention, replication in a cell can include theproduction of infectious viral particles, i.e., viral particles that caninfect a cell and result in the production of more infectious viralparticles.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded DNA and RNA. Apolynucleotide may include nucleotide sequences having differentfunctions, including for instance coding sequences, and non-codingsequences such as regulatory sequences and/or non-translated regions. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidecan be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. The term “heterologous polynucleotide”refers to a polynucleotide that has been inserted into the HCV genome,typically by using recombinant DNA techniques, and is not naturallyoccurring.

The terms “3′ non-translated RNA,” “3′ non-translated region,” and“3“untranslated region” are used interchangeably, and are terms of art.The term refers to the nucleotides that are at the 3′ end of thepositive-sense strand of the HCV polynucleotide, the complement thereof(i.e., the negative-sense RNA), and the corresponding DNA sequences ofthe positive-sense and the negative-sense RNA sequences. The 3′non-translated RNA includes, from 5′ to 3′, nucleotides of variablelength and sequence (referred to as the variable region), apoly-pyrimidine tract (the poly U-UC region), and a highly conservedsequence of about 100 nucleotides (the conserved region) (see FIG. 2).The variable region begins at the first nucleotide following the stopcodon of the NS5B coding region, and ends immediately before thenucleotides of the poly U-UC region. The poly U-UC region is a stretchof predominantly U residues, CU residues, or C(U)n-repeats. When thenucleotide sequence of a variable region is compared between members ofthe same genotype, there is typically a great deal of similarity;however, there is typically very little similarity in the nucleotidesequence of the variable regions between members of different genotypes(see, for instance, Yamada et al., Virology, 223, 255-261 (1996)). Thelength of the variable region can vary.

The terms “5′ non-translated RNA,” “5′ non-translated region,” “5′untranslated region” and “5′ noncoding region” are used interchangeably,and are terms of art (see Bukh et al., Proc. Nat. Acad. Sci. USA, 89,4942-4946 (1992)). The term refers to the nucleotides that are at the 5′end of the positive-sense strand of the HCV polynucleotide, thecomplement thereof (i.e., the negative-sense RNA), and the correspondingDNA sequences of the positive-sense and the negative-sense RNAsequences. The 5′ NTR includes about 341 nucleotides. The lastnucleotide of the 5′ NTR is immediately upstream and adjacent to thefirst nucleotide of the coding sequence encoding the hepatitis C viruspolyprotein.

A “coding region” or “coding sequence” is a nucleotide region thatencodes a polypeptide and, when placed under the control of appropriateregulatory sequences, expresses the encoded polypeptide. The boundariesof a coding region are generally determined by a translation start codonat its 5′ end and a translation stop codon at its 3′ end. A codingregion can encode one or more polypeptides. For instance, a codingregion can encode a polypeptide that is subsequently processed intoseveral polypeptides. A regulatory sequence or regulatory region is anucleotide sequence that regulates expression of a coding region towhich it is operably linked. Nonlimiting examples of regulatorysequences include promoters, transcription initiation sites, translationstart sites, internal ribosome entry sites, translation stop sites, andterminators. “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A regulatory sequence is “operablylinked” to a coding region when it is joined in such a way thatexpression of the coding region is achieved under conditions compatiblewith the regulatory sequence.

As used herein the term “marker” refers to a molecule, for instance, apolypeptide. A “selectable marker” is a polypeptide that inhibits acompound, for instance an antibiotic, from preventing cell growth. A“detectable marker” is a polypeptide that can be detected. A marker canbe both selectable and detectable.

“Polypeptide” as used herein refers to a polymer of amino acids and doesnot refer to a specific length of a polymer of amino acids. Thus, forexample, the terms peptide, oligopeptide, protein, and enzyme areincluded within the definition of polypeptide. This term also includespost-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like.

As used herein a “fusion polypeptide” refers to a polypeptide encoded bya coding region that is made up of two coding regions that have beenjoined together in frame, typically using recombinant DNA techniques,such that the two coding regions now encode a single polypeptide.

As used herein, a “transactivator” is a polypeptide that affects intrans the expression of a transactivated coding region. A“transactivated coding region” is a coding region to which is operablylinked an operator sequence. As used herein, the term “operatorsequence” is a type of regulatory region and includes a polynucleotidewith which a transactivator can interact to alter expression of anoperably linked transactivated coding region.

An “isolated” virus means a virus that has been removed from its naturalenvironment. For instance, a virus that has been removed from an animalis an isolated virus. Another example of an isolated virus is one thathas been removed from the cultured cells in which the virus waspropagated, for instance by removing media containing the virus. A virusof this invention may be purified, i.e., essentially free from any otherassociated cellular products or other impurities. The term “purified” isdefined as encompassing preparations of a virus having less than about50%, more preferable less than about 25% contaminating associatedcellular products or other impurities.

As used herein, the phrase “selecting a replication competent HCV RNA”refers to identifying a cell that includes a replication competent HCVRNA under conditions that prevent the replication of cells that do notinclude a replication competent HCV RNA.

A “hepatitis C virus polyprotein” refers to a polypeptide that ispost-translationally cleaved to yield more than one polypeptide. Unlessnoted otherwise, a hepatitis C virus polyprotein yields the polypeptidescore (also referred to as nucleocapsid), E1, E2, P7, NS2, NS3, NS4A,NS4B, NS5A, and NS5B. Optionally, a hepatitis C virus polyprotein alsoyields protein F (see Xu et al., EMBO J., 20, 3840-3848 (2001).

A “subgenomic” HCV polynucleotide, preferably an RNA, refers to an HCVRNA that does not include the entire HCV genome. A subgenomic HCV RNAtypically includes a coding region encoding only a portion of ahepatitis C virus polyprotein, e.g., the nucleotides encoding one ormore polypeptide is not present. Such a hepatitis C virus polyprotein isreferred to as a “subgenomic hepatitis C virus polyprotein.” In someaspects of the invention, an HCV RNA contains a subgenomic hepatitis Cvirus polyprotein that does not include polypeptides encoded by the 5′end of the hepatitis C virus polyprotein. Thus, a subgenomic hepatitis Cvirus polyprotein may encode the polypeptides NS3, NS4A, NS4B, NS5A, andNS5B; NS2, NS3, NS4A, NS4B, NS5A, and NS5B; P7, NS2, NS3, NS4A, NS4B,NS5A, and NS5B; E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B; or E1, E2,P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. In other aspects of theinvention, an HCV RNA contains a subgenomic hepatitis C viruspolyprotein that does not include polypeptides persent in an internalportion of a hepatitis C virus polyprotein. Thus, a subgenomic hepatitisC virus polyprotein may encode, for instance, the polypeptides NS3,NS4A, NS4B, and NS5B. Replication of a subgenomic HCV RNA in a cellincludes the synthesis of viral nucleic acid, for instance synthesis ofthe negative-sense strand, and typically does not include the productionof infectious viral particles.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Genomic organization of MK0-Z, ds-MK0-Z, and 3′ETZ. Therightward facing arrows, location and direction of transcriptioninitiation; 5′NTR, 5′ non-translated RNA; C, core protein; E1, envelopeprotein 1; E2, envelope protein 2; E2-p7, a polypeptide of about 7 kDa;NS2, non-structural protein 2; NS3, non-structural protein 3; NS4A,non-structural protein 4A; NS4B, non-structural protein 4B; NS5A,non-structural protein 5A; NS5B, non-structural protein 5B; EMCV IRES,encephalomyocarditis virus internal ribosome entry site; tat, portion ofthe human immunodeficiency virus I (HIV 1) tat protein; 2A, 2Aproteinase of foot-and-mouth disease virus (FMDV); Zeo, polypeptideencoding resistance to phleomycin; 3′NTR, 3′ non-translated RNA.

FIG. 2. Site of insertion of heterologous sequence within the 3′NTR (3′non-translated RNA) of H77C strain (pCV-H77C). Variable region, polyU-UC, and Conserved region refer to regions of the 3′ non-translatedRNA; EMCV IRES, tat, FMDV 2A, and Zeo, see legend to FIG. 1; NS5B refersto the last 12 nucleotides that encode NS5B.

FIG. 3. Schematic depicting release of SEAP from a reporter cell line byexpression of Tat from a modified HCV RNA. EMCV, tat, 2A, and Zeo, seelegend to FIG. 1; HIV-LTR, HIV I long terminal repeat transcriptionalregulator; SEAP, secretory alkaline phosphatase.

FIG. 4. SEAP activity in medium collected from cells followingtransfection with RNAs. (A) Huh7-SEAP-o10 cells. (B) Huh7-SEAP-N7 cells.The smaller graph A and B each depict days 1 and 6, but use differentscales. Mock, cells exposed to transfection conditions but not RNA;3′ETZ, MK0-Z, and dS-MK0-Z, the constructs shown in FIG. 1; y-axis,units of secretory alkaline phosphatase activity measured by luminescentsignal detected by a TD-20/20 Luminometer (Turner Design, Sunnyvale,Calif.).

FIG. 5. The passage history of two Huh-SEAP-o10 cell sublines (MK0-Z.C-Aand MK0-Z.C-B) that were infected with MK0-K and the secretory alkalinephosphatase (SEAP) activity in supernatant media collected atapproximately weekly intervals from both surviving cell lines. dSma(C-A) and dSma (C-B) are two Huh-SEAP-o10 cell sublines infected withsupernatant fluids collected from cells transfected in parallel withdS-MK0-Z (NS5B-deletion mutant) RNA. Split, points at which the cultureswere split are indicated by arrows. The top panel shows the timing andmagnitude of Zeocin selection pressure (top panel, mg/ml).

FIG. 6. SEAP expression profiles of Huh-SEAP-o10 cells. (A) AbsoluteSEAP activities of supernatant media from cells inoculated withsupernatant fluids of C-A and C-B MK0-Z infected cell lines. “I1”inoculum=media from C-A subline, “I4” inoculum=media from C-B subline.None=mock infections. (B) SEAP activity relative to SEAP activity ofmock-infected control Huh-SEAP-o10 cells (lost during Zeocin selection).

FIG. 7. LightCycler RT-PCR detection of viral RNA in supernatant fluidsof C-A and C-B cells. The plot demonstrates the melting curves of thefluorescence resonance energy transfer signal from products generatedfrom the cell culture samples and associated controls. Fluorescence−d[F2/F1]/dT, the melting curve as calculated by the LightCycler thermalcycler.

FIG. 8. TaqMan RT-PCR detection of HCV RNA in C-A and C-B cell culturesupernat ants.

FIG. 9. Nucleotide sequence of MK0-Z (SEQ ID NO:17). The initiationcodon of the viral polyprotein which undergoes post-translationalcleavage is the ATG at nucleotides 342-344. The initiation codon of theinserted heterologous polynucleotide is the ATG at nucleotides9907-9909.

FIG. 10. Nucleotides 342-10,803 of SEQ ID NO:17, and the polyprotein(SEQ ID NO:20). The amino acid sequences SEQ ID NO:32, SEQ ID NO:33, andSEQ ID NO:34 encoded by nucleotides 9,390-9,485, nucleotides9,489-9,794, and nucleotides 9,798-9,887 of SEQ ID NO:17, respectively,are shown. The amino acid sequence (SEQ ID NO:21) encoded by theheterologous polynucleotide (i.e., nucleotides 9907-10,602 of SEQ IDNO:17) is also shown.

FIG. 11. The results of Taqman RT-PCR of a chimpanzee inoculated withMK0-Z RNA. The term ge/ml refers to genomic equivalents per milliliter.

FIG. 12. Nucleotide sequence of HIVSEAP (SEQ ID NO:18). The HIV longterminal repeat (LTR) is depicted at nucleotides 1-719, and secretoryalkaline phosphatase is encoded by the nucleotides 748-2239.

FIG. 13. (A) Organization of the subgenomic HCV RNA replicons. Openreading frames are depicted as boxes, and untranslated segments of thedicistronic RNAs are depicted as solid lines. The sequence of BNeo/3-5B(shaded box) is identical to that of 1377NS3-3/wt, described previouslyby Lohmann et al. (Science, 285, 10-113 (1999)). NNeo/3-5B containsmostly HCV-N-derived sequence (open boxes). The amino acid sequence ofNS3 in NNeo/3-5B differs from that of HCV-N at only 2 amino acidresidues while the 5′- and 3′ UTR sequences are identical. “ΔC”indicates the N-terminal segment of the HCV core protein that isexpressed as a fusion with Neo in these replicons. (B) Locations of theS2205I and R2889G BNeo/3-5B-adaptive mutations and the MLVNGDDLVVdeletion introduced into the replicons shown in panel A.

FIG. 14. Organization of selectable dicistronic RNAs containing HCV-Nsequence encoding NS2, the envelope proteins E1 and E2, and/or the coreprotein within the 3′ cistron. NTR, nontranslated region.

FIG. 15. Alignment of the amino acid sequences of the NS5A proteinsencoded by NNeo/3-5B and Neo/3-5B. The ISDR is shaded, with the4-amino-acid SSYN insertion in NNeo/3-5B shown in boldface type andenclosed in a box. Arrows indicate the location of single-basesubstitutions and insertions and the large 47-amino-acid deletion thathas been shown previously to enhance the replication capacity ofBNeo/3-5B (Blight et al., Science, 290, 1972-1974 (2000), Krieger etal., J. Virol., 75, 4614-4624 (2001), Lohmann et al., J. Virol., 75,1437-1449 (2002)). The asterisk indicates the S20051 mutation.

FIG. 16. Enzyme reporter system. (A) Organization of pEt2AN. A solidsquare represents the CMV promoter region; a solid arrow the T7promoter; a thick line the EMCV IRES and the open box for the openreading frame encoding the fusion polypeptide tat-2A-Neo. (B) SEAPexpression following pEt2AN DNA transfection into En5-3 cells (▴). Theexpression of tat from this plasmid is dependent on the CMV promoter.Note that SEAP activity is reported in arbitrary units. SEAP expressionfrom En5-3 cells without DNA transfection was also shown (▪). (C) SEAPexpression following electroporation of En5-3 cells with RNA transcribedin vitro from pEt2AN (▴). SEAP expression from En5-3 cells without RNAtransfection was also shown (▪).

FIG. 17. (A) Organization of subgenomic HCV RNA replicons encoding tat.Open reading frames are depicted as boxes, and nontranslated segments ofthe dicistronic RNAs as solid lines. ΔC indicates the N-terminal 14amino acid core protein segment. (B) Additional mutations engineeredinto the replicons.

FIG. 18. (A) Product of in vitro translation reactions programmed withthe indicated RNAs. (*) indicates the expected positions of the majorprotein products anticipated to be produced from the dicistronic RNAs.(B) SEAP activity present in tissue culture media 72 hrs followingtransient transfection with synthetic RNAs transcribed from theindicated plasmids.

FIG. 19. (A) Northern Blot analysis of replicon RNAs following passageof stable G418-resistant cell clones. (B) HCV RNA abundance detected byTaqMan RT-PCR, normalized to a total cellular RNA standard, andpresented as copies of HCV RNA per pg total cellular RNA. The same RNAsamples were used as in northern blot analysis in FIG. 19A. Open barrepresents BΔCtat2ANeo(SI), solid bar represents Btat2ANeo(SI); graybar, for Ntat2ANeo(RG).

FIG. 20. (A) SEAP activity present in supernatant culture media atvarious time point following passage of stable cell lines. Btat2ANeo(SI)(▴), Ntat2ANeo(RG) (▪), BACtat2ANeo(SI) (●), En5-3 (⋄). Bars show therange of SEAP activity from duplicate experiments. (B) Linear regressionanalysis of SEAP activity vs. abundance of replicon RNA in the culture,as determined by densitometry of northern blots. Btat2ANeo(SI) (▴- - -), Ntat2ANeo(RG) (▪- - - ).

FIG. 21. SEAP activity following transient transfection of En5-3 cellswith (A) Btat2Aneo and (B) Ntat2Aneo with various mutations. Wt(∘), SI(▪), RG (▴), AGDD (X), N-Δ5ASI (*). Arrow indicates trypsinization andpassage of cells.

FIG. 22. Suppression of HCV replicon amplification by interferon-α2b.(A) SEAP activity secreted from cells supporting replication ofBtat2ANeo(SI) over successive 24 hr intervals following addition ofinterferon to the medium. (B) SEAP secretion from Ntat2ANeo(RG) cells.Interferon concentrations were: (*) 100 units/ml; (X) 10 units/ml; (▴) 1unit/ml; (▪) no interferon. SEAP expression from En5-3 cells withoutinterferon treatment was also shown (♦). SEAP expression from En5-3cells was not affected by interferon treatment.

FIG. 23. Suppression of HCV replicon RNA abundance by interferon-α2b inthe cell cultures depicted in FIG. 22. (A) Intracellular abundance ofHCV RNA in cells supporting replication of Btat2ANeo(SI) at 24, 72 and120 hrs following addition of interferon to the medium. (B) RNAabundance in Ntat2ANeo(RG) cells under similar conditions. HCV RNA wasquantified by RT-PCR analysis, and normalized to a total cellular RNAstandard (see legend to FIG. 19B). Interferon concentrations were: (*)100 units/ml; (X) 10 units/ml; (▴) 1 unit/ml; (▪) no interferon.

FIG. 24. Nucleotide sequences of constructs described in FIG. 17. Thenucleotide sequence of the 5′ NTR is disclosed at SEQ ID NO:35, thenucleotide sequence of the ΔCtat2ANeo is disclosed at SEQ ID NO:36, thenucleotide sequence of the tat2ANeo is disclosed at SEQ ID NO:37, thenucleotide sequence of the EMCV IRES located between the two cistrons isdisclosed at SEQ ID NO:38. The nucleotide sequence encoding hepatitis Cvirus polyprotein derived from HCV-N is disclosed at SEQ ID NO:39, andthe amino acid sequence (SEQ ID NO:40) of the polyprotein encoded by thenucleotides 2077-11121 is also shown. The nucleotide sequence encodinghepatitis C virus polyprotein derived from Con1 is disclosed at SEQ IDNO:41, and the amino acid sequence (SEQ ID NO:42) of the polyproteinencoded by the nucleotides 2119-8073 is also shown. The nucleotidesequence of the 3NTR that is present in those replicons having anhepatitis C virus polyprotein derived from HCV-N is disclosed atnucleotides 11122-11349 of SEQ ID NO:39. The nucleotide sequence of the3′NTR that is present in those replicons having an hepatitis C viruspolyprotein derived from Con1 is disclosed at nucleotides 8074-8307 ofSEQ ID NO:41.

DETAILED DESCRIPTION OF THE INVENTION

Hepatitis C Virus

The present invention provides HCV polynucleotides, preferably RNA, thatinclude a heterologous polynucleotide. In some aspects of the invention,the HCV includes a coding sequence encoding an hepatitis C viruspolyprotein, and in other aspects the HCV includes a coding regionencoding a portion of an HCV polyprotein. Preferably, the HCV arereplication competent. Preferably the HCV are isolated, more preferably,purified. Unless otherwise noted, HCV polynucleotide, and other termsthat refer to all or a part of an HCV polynucleotide (including, forinstance, “3“non-translated RNA”) include an RNA sequence of thepositive-sense genome RNA, the complement thereof (i.e., thenegative-sense RNA), and the DNA sequences corresponding to thepositive-sense and the negative-sense RNA sequences.

It is expected that HCV polynucleotides from different sources,including molecularly cloned laboratory strains, for instance cDNAclones of HCV, and clinical isolates can be used in the methodsdescribed below to yield replication competent HCV of the presentinvention. Examples of molecularly cloned laboratory strains include theHCV that is encoded by pCV-H77C (Yanagi et al., Proc. Natl. Acad. Sci.,USA, 94, 8738-8743 (1997)), and pHCV-N as modified by Beard et al.(Hepatol., 30, 316-324 (1999)). Clinical isolates can be from a sourceof infectious HCV, including tissue samples, for instance from blood,plasma, serum, liver biopsy, or leukocytes, from an infected animal,including a human or a primate.

It is expected that the HCV polynucleotides of the present invention arenot limited to a specific genotype. For instance, an HCV of the presentinvention can be genotype 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 4, 5a, or 6a(as defined by Simmons, Hepatology, 21, 570-583 (1995)). It is alsoexpected that HCV used in the methods described below can be prepared byrecombinant, enzymatic, or chemical techniques. In some aspects, an HCVthat is modified as described herein to include a heterologouspolynucleotide is able to replicate in vivo, preferably in a chimpanzee,prior to inserting the heterologous polypeptide. Methods for determiningwhether an HCV is able to replicate in a chimpanzee are describedherein.

In some aspects of the present invention, the nucleotide sequence of anHCV polynucleotide used in the methods of the present invention issimilar to the nucleotide sequence of an HCV, preferable an HCV ofgenotype 1a, 1b, 2a, or 2b. An example of an HCV of genotype 1a ispresent at Genbank accession AF011751. Examples of an HCV of genotype 1bare present at Genbank accession AF139594, Genbank accession AJ238799,or the sequences present at FIG. 24. An example of an HCV of genotype 2ais present at Genbank accession AF238481. An example of an HCV ofgenotype 2b is present at Genbank accession AB030907. The similarity isreferred to as structural similarity and may be determined by aligningthe residues of the two polynucleotides (i.e., the nucleotide sequenceof a candidate nucleotide sequence and the nucleotide sequence of HCV,or a portion thereof) to optimize the number of identical nucleotidesalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofshared nucleotides, although the nucleotides in each sequence mustnonetheless remain in their proper order. A candidate nucleotidesequence is the nucleolide sequence being compared to the nucleotidesequence of the HCV, or a portion thereof. Two nucleotide sequences canbe compared using standard software algorithms. Preferably, twonucleotide sequences are compared using the Blastn program of the BLAST2 search algorithm, as described by Tatusova, et al. (FEMS MicrobiolLeft 1999, 174:247-250), and available atncbi.nim.nih.gov/gorf/bl2.html. Preferably, the default values for allBLAST 2 search parameters are used, including reward for match=1,penalty for mismatch=−2, open gap penalty=5, extension gap penalty=2,gap x_dropoff=50, expect=10, wordsize=11, and filter on. In thecomparison of two nucleotide sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, apolynucleotide includes a nucleotide sequence having a structuralsimilarity with the coding region of an HCV, or a portion thereof, of atleast about 66%, at least about 77%, at least about 91%, at least about94%, at least about 96%, or at least about 99% identity.

Specific mutations increasing the replicative capacity of HCVpolynucleotides have been characterized for HCV 1b subgenomic RNAreplicons (see, for instance, Blight et al., Science, 290, 1972-1975(2000); Lohmann et al., “Adaptation of selectable HCV replicon to ahuman hepatoma cell line,” Abstract P038, 7th International Meeting onHepatitis C virus and Related viruses (Molecular Virology andPathogenesis), The Marriott resort Hotel, Gold coast, Queensland,Australia, December 3-7 (2000); and Guo et al., “Identification of anovel RNA species in cell lines expressing HCV subgenomic replicons,”Abstract P045, 7th International Meeting on Hepatitis C virus andRelated viruses (Molecular Virology and Pathogenesis), The Marriottresort Hotel, Gold coast, Queensland, Australia, December 3-7 (2000)).Such mutations are referred to herein as “cell culture adaptivemutations.” It is expected that the introduction of these individualmutations may enhance the replication capacity of an HCV of some aspectsof the present invention. The approximate locations and types of somemutations are shown in Table 1. The precise location of these cellculture adaptive mutations can vary between members of differentgenotypes, and between members of the same genotype. For instance, withmutations 2442 and 2884 listed in Table 1, in HCV genotype 1a thelocations of these mutations are 2443 and 2885, respectively. Thelocation of a mutation introduced into an HCV of the present inventionto enhance replication is expected to be within 4 amino acids,preferably within 3 amino acids, more preferably within 2 amino acids,most preferably within 1 amino acid of the positions listed in Table 1.Another example of an adaptive mutation of HCV-N is the insertion ofamino acids SSYN present at position 2220-2223. TABLE 1 Adaptivemutations in an HCV of genotype 1b. Amino acid position¹ Mutation² 1202E to G 1281 T to I 1283 R to G 1383 E to A 1577 K to R 1609 K to E 1757L to I 1936 P to S 2163 E to G 2177 D to H, or D to N 2189 R to G 2196 Pto S 2197 S to P, or S to C 2199 A to S, or A to T 2201 deletion of S2204³ S to I 2207-2254 Deletion of 48 amino acids 2330 K to E 2442 I toV 2884⁴ R to G¹Amino acid position refers to amino acid number where the first aminoacid is the first amino acid of the polyprotein expressed by the HCV atGenbank Accession number AJ238799.²Amino acids are listed in the single letter code. The first amino acidis the wild-type amino acid, and the second amino acid is the residuepresent in the mutant.³Amino acid 2205 in the polyprotein expressed by the HCV at GenbankAccession number AF139594.⁴Amino acid 2889 in the polyprotein expressed by the HCV at GenbankAccession number AF139594.

Cell culture adaptive mutations can be introduced into an HCVpolynucleotide of the present invention by mutagenesis of the nucleotidesequence of the HCV in the form of plasmid DNA. Methods for targetedmutagenesis of nucleotide sequences are known to the art, and include,for instance, PCR mutagenesis.

In some aspects of the invention, the heterologous polynucleotide ispresent in the HCV 3′ non-translated RNA, for instance, in the variableregion of the 3′ non-translated RNA. In some aspects of the invention,the heterologous polynucleotide is inserted into the variable regionsuch that the variable region is not removed. Alternatively, deletionsof the variable region can be made, in whole or in part, and replacedwith the heterologous polynucleotide. Preferably, in some aspects of theinvention, when the HCV has the genotype 1a, more preferably, the strainH77C, the heterologous polynucleotide is inserted in the variable regionbetween nucleotides 5 and 6 of the sequence 5′CUCUUAAGC 3′, where thesequence shown corresponds to the positive-strand.

A heterologous polynucleotide can include a non-coding region and/or acoding region, preferably a coding region. The coding region can encodea polypeptide including, for instance, a marker, including a detectablemarker and/or a selectable marker. Examples of detectable markersinclude secretory alkaline phosphatase, green fluorescent protein, andmolecules that can be detected by antibody. Examples of selectablemarkers include molecules that confer resistance to antibiotics,including the antibiotics kanamycin, ampicillin, chloramphenicol,tetracycline, neomycin, and formulations of phleomycin D1 including, forexample, the formulation available under the trade-name ZEOCIN(Invitrogen, Carlsbad, Calif.). Other examples of polypeptides that canbe encoded by the coding region include a transactivator, and/or afusion polypeptide. Preferably, when the polypeptide is a fusionpolypeptide, the coding region includes nucleotides encoding a marker,more preferably, nucleotides encoding a fusion between a transactivatorand a marker. Optionally, the coding region can encode an immunogenicpolypeptide. When the heterologous polynucleotide includes a codingregion, the HCV is typically dicistronic, i.e., the coding region of theheterologous polynucleotide and the coding region encoding the HCVpolyprotein or portion thereof are separate.

An “immunogenic polypeptide” refers to a polypeptide which elicits animmunological response in an animal. An immunological response to apolypeptide is the development in a subject of a cellular and/orantibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directedspecifically to an epitope or epitopes of the polypeptide fragment.

A transactivator is a polypeptide that affects in trans the expressionof a coding region, preferably a coding region integrated in the genomicDNA of a cell. Such coding regions are referred to herein as“transactivated coding regions.” The cells containing transactivatedcoding regions are described in detail herein in the section “Methods ofuse.” Transactivators useful in the present invention include those thatcan interact with a regulatory region, preferably an operator sequence,that is operably linked to a transactivated coding region. As usedherein, the term “transactivator” includes polypeptides that interactwith an operator sequence and either prevent transcription frominitiating at, activate transcription initiation from, or stabilize atranscript from, a transactivated coding region operably linked to theoperator sequence. Examples of useful transactivators include the HIVtat polypeptide (see, for example, the polypeptide SEQ ID NO:19,MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQPRGDPTGPKE which is encoded by nucleotides 5377to 5591 and 7925 to 7970 of Genbank accession number AF033819), andMEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQPTSQSRGDPTGPKE, the polypeptide present at aminoacids 4-89 of SEQ ID NO:21. The HIV tat polypeptide interacts with theHIV long terminal repeat. Other useful transactivators include human Tcell leukemia virus tax polypeptide (which binds to the operatorsequence tax response element, Fujisawa et al., J. Virol., 65, 4525-4528(1991)), and transactivating polypeptides encoded by spumaviruses in theregion between env and the LTR, such as the bel-1 polypeptide in thecase of human foamy virus (which binds to the U3 domain of theseviruses, Rethwilm et al., Proc. Natl. Acad. Sci. USA, 88, 941-945(1991)). Alternatively, a post-transcriptional transactivator, such asHIV rev, can be used. HIV rev binds to a 234 nucleotide RNA sequence inthe env gene (the rev-response element, or RRE) of HIV(Hadzopolou-Cladaras et al., J. Virol., 63, 1265-1274 (1989)).

Other transactivators that can be used are those having similarity withthe amino acid sequence of SEQ ID NO:19 or amino acids 4-89 of SEQ IDNO:21. The similarity is referred to as structural similarity and isgenerally determined by aligning the residues of the two amino acidsequences (i.e., a candidate amino acid sequence and the amino acidsequence of SEQ ID NO:19 or amino acids 4-89 of SEQ ID NO:21) tooptimize the number of identical amino acids along the lengths of theirsequences; gaps in either or both sequences are permitted in making thealignment in order to optimize the number of identical amino acids,although the amino acids in each sequence must nonetheless remain intheir proper order. A candidate amino acid sequence is the amino acidsequence being compared to an amino acid sequence present in SEQ IDNO:19 or amino acids 4-89 of SEQ ID NO:21. A candidate amino acidsequence can be isolated from a virus, or can be produced usingrecombinant techniques, or chemically or enzymatically synthesized.Preferably, two amino acid sequences are compared using the Blastpprogram of the BLAST 2 search algorithm, as described by Tatusova, etal. (FEMS Microbiol Lett 1999, 174: 247-250), and available atwww.ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, the default values forall BLAST 2 search parameters are used, including matrix=BLOSUM62; opengap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10,wordsize=3, and filter on. In the comparison of two amino acid sequencesusing the BLAST search algorithm, structural similarity is referred toas “identities.” Preferably, a transactivator includes an amino acidsequence having a structural similarity with SEQ ID NO:19 or amino acids4-89 of SEQ ID NO:21 of at least about 70%, at least about 80%, at leastabout 90%, at least about 94%, at least about 96%, or at least about 99%identity. Typically, an amino acid sequence having a structuralsimilarity with SEQ ID NO:19 or amino acids 4-89 of SEQ ID NO:21 has tatactivity. Whether such a polypeptide has activity can be evaluated bydetermining if the amino acid sequence can interact with an HIV LTR,preferably, alter transcription from a coding sequence operably linkedto an HIV LTR.

Active analogs or active fragments of a transactivator can be used inthe invention. An active analog or active fragment of a transactivatoris one that is able to interact with an operator sequence and eitherprevent transcription from initiating at, activate transcriptioninitiation from, or stabilize a transcript from, a transactivated codingregion operably linked to the operator sequence.

Active analogs of a transactivator include polypeptides havingconservative amino acid substitutions that do not eliminate the abilityto interact with an operator and alter transcription. Substitutes for anamino acid may be selected from other members of the class to which theamino acid belongs. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, aspartate, and glutamate. Thepositively charged (basic) amino acids include arginine, lysine, andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of preferred conservative substitutionsinclude Lys for Arg and vice versa to maintain a positive charge; Glufor Asp and vice versa to maintain a negative charge; Ser for Thr sothat a free —OH is maintained; and Gin for Asn to maintain a free NH₂.

Active fragments of a transactivator include a portion of thetransactivator containing deletions or additions of about 1, about 2,about 3, about 4, or at least about 5 contiguous or noncontiguous aminoacids such that the resulting transactivator will alter expression of anoperably linked transactivated coding region. A preferred example of anactive fragment of the HIV tat polypeptide includes amino acids aminoacids 1-48 of SEQ ID NO:19, or amino acids 4-51 of SEQ ID NO:21.

In those aspects of the invention where the heterologous polynucleotideincludes a coding region that encodes a fusion polypeptide, the fusionpolypeptide can further include amino acids corresponding to acis-active proteinase. When the fusion polypeptide is a fusion between atransactivator and a marker, preferably the fusion polypeptide alsoincludes amino acids corresponding to a cis-active proteinase.Preferably the amino acids corresponding to a cis-active proteinase arepresent between the amino acids corresponding to the transactivator andthe marker. A cis-active proteinase in this position allows the aminoacids corresponding to the transactivator and the marker to bephysically separate from each other in the cell within which the HCV ispresent. Examples of cis-active proteinases that are useful in thepresent invention include the cis-active 2A proteinase of foot-and-mouthdisease (FMDV) virus (see, for example, U.S. Pat. No. 5,846,767 (Halpinet al.) and U.S. Pat. No. 5,912,167 (Palmenberg et al.)), ubiquitin(see, for example, Tauz et al., Virology, 197, 74-85 (1993)), and theNS3 recognition site GADTEDVVCCSMSY (SEQ ID NO:31) (see, for example,Lai et al., J. Virol., 74, 6339-6347 (2000)).

Active analogs and active fragments of cis-active proteinases can alsobe used. Active analogs of a cis-acting proteinase include polypeptideshaving conservative amino acid substitutions that do not eliminate theability of the proteinase to catalyze cleavage. Active fragments of acis-active proteinase include a portion of the cis-active proteinasecontaining deletions or additions of one or more contiguous ornoncontiguous amino acids such that the resulting cis-active proteinasewill catalyze the cleavage of the proteinase.

In some aspects of the invention, the heterologous polynucleotide mayfurther include a regulatory region that is operably linked to thecoding region of the heterologous polynucleotide. Preferably, aregulatory region located 5′ of the operably linked coding regionprovides for the translation of the coding region.

A preferred regulatory region located 5′ of an operably linked codingregion is an internal ribosome entry site (IRES). An IRES allows aribosome access to mRNA without a requirement for cap recognition andsubsequent scanning to the initiator AUG (Pelletier, et al., Nature,334, 320-325 (1988)). An IRES is located upstream of the translationinitiation codon, e.g., ATG or AUG, of the coding sequence to which theIRES is operably linked. The distance between the IRES and theinitiation codon is dependent on the type or IRES used, and is known tothe art. For instance, poliovirus IRES initiates a ribosometranslocation/scanning process to a downstream AUG codon. For other IRESelements, the initiator codon is generally located at the 3′ end of theIRES sequence. Examples of an IRES that can be used in the inventioninclude a viral IRES, preferably a picomaviral IRES or a flaviviralIRES. Examples of poliovirus IRES elements include, for instance,poliovirus IRES, encephalomyocarditis virus IRES, or hepatitis A virusIRES. Examples of preferred flaviviral IRES elements include hepatitis Cvirus IRES, GB virus B IRES, or a pestivirus IRES, including but notlimited to bovine viral diarrhea virus IRES or classical swine fevervirus IRES. Other IRES elements with similar secondary and tertiarystructure and translation initiation activity can either be generated bymutation of these viral sequences, by cloning of analogous sequencesfrom other viruses (including picornaviruses), or prepared by enzymaticsynthesis techniques.

The size of the heterologous polynucleotide is not critical to theinvention. It is expected there is no lower limit on the size of theheterologous polynucleotide. It is expected that there is an upper limiton the size of the heterologous polynucleotide. This upper limit can beeasily determined by a person skilled in the art, as heterologouspolynucleotides that are greater than this upper limit adversely affectreplication of an HCV polynucleotide. In increasing order of preference,the heterologous polynucleotide is at least about 10 nucleotides, atleast about 20 nucleotides, at least about 30 nucleotides, mostpreferably at least about 40 nucleotides.

In some aspects of the invention, the heterologous polynucleotide ispresent in an HCV downstream of the 5′ NTR. For instance, the firstnucleotide of the heterologous polynucleolide may be immediatelydownstream and adjacent to the last nucleotide of the 5′ NTR.Alternatively, the first nucleotide of the heterologous polynucleotidemay be about 33 to about 51 nucleotides, more preferably, about 36 toabout 48 nucleotides, downstream of the last nucleotide of the 5′ NTR.Typically, when the first nucleotide of the heterologous polynucleotideis not immediately downstream of the last nucleotide of the 5′ NTR, thenucleotides in between the 5′ NTR and the heterologous polynucleotideencode the amino terminal amino acids of the HCV core polypeptide.

In those aspects of the invention where the heterologous polynucleotidepresent in an HCV is inserted downstream of the 5′ NTR and upstream ofthe coding region encoding the HCV polyprotein or a portion thereof, theheterologous polynucleotide typically includes a regulatory regionoperably linked to the downstream coding region. Preferably, theregulatory region provides for the translation of the downstream codingregion. The size of the regulatory region may be from about 400nucleotides to about 800 nucleotide, more preferably, about 600nucleotides to about 700 nucleotides. Preferably, the regulatory regionis an IRES. Examples of IRES elements are described herein.

In those aspects of the invention where the HCV polynucleotide includesa portion of the hepatitis C virus polyprotein, the 5′ end of the codingregion encoding the HCV polyprotein may further include about 33 toabout 51 nucleotides, more preferably, about 36 to about 48 nucleotides,that encode the first about 11 to about 17, more preferably, about 12 toabout 16, amino acids of the core polypeptide. The result is a fusionpolypeptide between the amino terminal amino acids of the corepolypeptide and the first polypeptide encoded by the heterologouspolnucleotide.

The replication competent HCV polynucleotide of the invention can bepresent in a vector. When a replication competent HCV is present in avector the HCV is DNA, including the 5′ non-translated RNA and the 3′non-translated RNA. Methods for cloning an HCV and inserting it into avector are known to the art (see, e.g., Yanagi et al., Proc. Natl. Acad.Sci., USA, 94, 8738-8743 (1997); and Rice et al., (U.S. Pat. No.6,127,116)). Such constructs are often referred to as molecularly clonedlaboratory strains, and an HCV that is inserted into a vector istypically referred to as a cDNA clone of the HCV. If the RNA encoded bythe HCV is able to replicate in vivo, the HCV present in the vector isreferred to as an infectious cDNA clone. A vector is a replicatingpolynucleotide, such as a plasmid, phage, cosmid, or artificialchromosome to which another polynucleotide may be attached so as tobring about the replication of the attached polynucleotide. A vector canprovide for further cloning (amplification of the polynucleotide), i.e.,a cloning vector, or for expression of the polypeptide encoded by thecoding region, i.e., an expression vector. The term vector includes, butis not limited to, plasmid vectors, viral vectors, cosmid vectors, orartificial chromosome vectors. Preferably the vector is a plasmid.Preferably the vector is able to replicate in a prokaryotic host cell,for instance Escherichia coli. Preferably, the vector can integrate inthe genomic DNA of a eukaryotic cell.

An expression vector optionally includes regulatory sequences operablylinked to the HCV such that the HCV is transcribed to produce RNAmolecules. These RNA molecules can be used, for instance, forintroducing an HCV to a cell that is in an animal or growing in culture.The terms “introduce” and “introducing” refer to providing an HCV to acell under conditions that the HCV is taken up by the cell in such a waythat the HCV can then replicate. The HCV can be a virus particle, or anucleic acid molecule, preferably RNA. The invention is not limited bythe use of any particular promoter, and a wide variety are known.Promoters act as regulatory signals that bind RNA polymerase in a cellto initiate transcription of a downstream (3′ direction) HCV. Thepromoter used in the invention can be a constitutive or an induciblepromoter. A preferred promoter for the production of HCV is T7 promoter.

Preferred examples of HCV polynucleotide of the present invention areshown in FIGS. 9, 10, and 17. It should be noted that while thesesequences are DNA sequences, the present invention contemplates thecorresponding RNA sequence, and RNA and DNA complements thereof, aswell.

Methods of Use

The present invention is directed to methods for identifying areplication competent HCV polynucleotide, including detecting and/orselecting for cells containing a replication competent HCVpolynucleotide. Typically, the cells used in this aspect of theinvention are cells growing in culture. Useful cultured cells willsupport the replication of the HCV of the present invention, and includeprimary human or chimpanzee hepatocytes, peripheral mononuclear cells,cultured human lymphoid cell lines (for instance lines expressing B-celland T-cell markers such as Bjab and Molt-4 cells), and continuous celllines derived from such cells, including Huh-7, HepG2, and PH5CH-8. Thecells may be primate or human cells, preferably human cells. In general,useful cells include those that support replication of HCV RNA,including, for instance, replication of the HCV encoded by pCV-H₇₇C, orreplication of the HCV encoded by pHCV-N as modified by Beard et al.(Hepatol., 30, 316-324 (1999)). A preferred cultured cell is HuH-7,which is known to workers in the field of HCV (see, for instance,Lohmann et al., Science, 285, 570-574 (1999)).

In some aspects of the invention, the cultured cell includes apolynucleotide that includes a coding region, the expression of which iscontrolled by a transactivator. Such a coding region is referred toherein as a transactivated coding region. A transactivated coding regionencodes a marker, preferably a detectable marker, for example, secretoryalkaline phosphatase. In some aspects of the invention, the detectablemarker is secretory alkaline phospahtase (SEAP). An example of an SEAPis encoded by nucleotides 748-2239 of SEQ ID NO:18. Typically, acultured cell that includes a polynucleotide having a transactivatedcoding region is used in conjunction with an HCV polynucleotide thatincludes a coding region encoding a transactivator.

The polynucleotide that includes the transactivated coding region can bepresent integrated into the genomic DNA of the cell, or present as partof a vector that is not integrated. Preferably, the polynucleotide isintegrated into the genomic DNA of the cell. Methods of modifying a cellto contain an integrated DNA are known to the art. An example of makingsuch a cell is described in Example 3 and Example 9.

Operably linked to the transactivated coding region is an operatorsequence. The interaction of a transactivator can alter transcription ofthe operably linked transactivated coding region. In those aspects ofthe invention where a transactivator increases transcription, preferablythere is low transcription of the transactivated coding region in theabsence of a transactivator, more preferably, essentially notranscription. An operator sequence can be present upstream (5′) ordownstream (3′) of a transactivated coding region. An operator sequencecan be a promoter, or can be a nucleotide sequence that is present inaddition to a promoter.

In some aspects of the invention, the operator sequence that is operablylinked to a transactivated coding sequence is an HIV long terminalrepeat (LTR). An example of an HIV LTR is depicted at nucleotides 1-719of SEQ ID NO:18. Also included in the present invention are operatorsequences having similarity to nucleotides 1-719 of SEQ ID NO:18. Thesimilarity between two nucleotides sequences may be determined asdescribed above, however, the candidate nucleotide sequence is comparedto the nucleotides 1-719 of SEQ ID NO:18. Preferably, an operatorsequence includes a nucleotide sequence having a structural similaritywith the nucleotides 1-719 of SEQ ID NO:18 of at least about 80%, morepreferably at least about 90%, most preferably at least about 95%identity. Typically, an operator sequence having structural similaritywith the nucleotides 1-719 of SEQ ID NO:18 has transcriptional activity.Whether such an operator sequence has transcriptional activity can bedetermined by evaluating the ability of the operator sequence to altertranscription of an operably linked coding sequence in response to thepresence of a polypeptide having tat activity, preferably, a polypeptideincluding the amino acids of SEQ ID NO:19 or amino acids 4-89 of SEQ IDNO:21.

In some aspects of the present invention, the replication of culturedcells may be inhibited by a selecting agent. Examples of selectingagents include antibiotics, including kanamycin, ampicillin,chloramphenicol, tetracycline, neomycin, and formulations of phleomycinD1. A selecting agent can act to prevent replication of a cell while theagent is present and the cell does not express a molecule that providesresistance to the selecting agent. Alternatively and preferably, aselecting agent can act to kill a cell that does not express a moleculethat provides resistance to the selecting agent. Typically, the moleculeproviding resistance to a selecting agent is expressed in the cell by anHCV polynucleotide of the present invention. Alternatively, the moleculeproviding resistance to a selecting agent is expressed by the cell butthe expression of the molecule is controlled by an HCV polynucleotide ofthe present invention that is present in the cell. The concentration ofthe selecting agent is typically chosen such that a cell that does notcontain a molecule providing resistance to a selecting agent does notreplicate. The appropriate concentration of a selecting agent variesdepending on the particular selecting agent, and can be easilydetermined by one having ordinary skill in the art using knowntechniques.

When a polynucleotide that includes a replication competent HCVpolynucleotide is introduced into a cell that is growing in culture, thepolynucleotide can be introduced using techniques known to the art. Suchtechniques include, for instance, liposome and non-liposome mediatedtransfection. The Examples describe the use of one type of liposomemediated transfection. Non-liposome mediated transfection methodsinclude, for instance, electroporation.

In some aspects of the invention, when a replication competent HCVpolynucleotide is identified using cultured cells, its ability toreplicate may be verified by introducing the HCV to a cell present in ananimal, preferably a chimpanzee. When the cell is present in the body ofan animal, the polynucleotide that includes a replication competent HCVcan be introduced by, for instance, subcutaneous, intramuscular,intraperitoneal, intravenous, or percutaneous intrahepaticadministration, preferably by percutaneous intrahepatic administration.Methods for determining whether an HCV polynucleotide is able toreplicate in a chimpanzee are known to the art (see, for example, Yanagiet al., Proc. Natl. Acad. Sci. USA, 94, 8738-8743 (1997), and Example2). In general, the demonstration of infectivity is based on theappearance of the virus in the circulation (blood) of the chimpanzeeover the days and weeks following the intrahepatic injection of the HCV.The presence of the virus can be confirmed by reversetranscription-polymerase chain reaction (RT-PCR) detection of the viralRNA, by inoculation of a second chimpanzee with transfer of thehepatitis C virus infection as indicated by the appearance of liverdisease and seroconversion to hepatitis C virus in ELISA tests, orpossibly by the immunologic detection of components of the hepatitis Cvirus (e.g., the core protein) in the circulation of the inoculatedanimal. It should be noted that seroconversion by itself would not be auseful indicator of infection in an animal injected with a viral RNAproduced using a molecularly cloned laboratory strain, as this RNA mayhave immunizing properties and be capable of inducing HCV-specificantibodies to proteins translated from an input RNA that isnon-replicating. Similarly, the absence of seroconversion does notexclude the possibility of viral replication and infection of achimpanzee with HCV.

Whether an HCV polynucleotide of the present invention is replicationcompetent can be determined using methods known to the art, includingmethods that use nucleic acid amplification to detect the result ofincreased levels of HCV replication. In some aspects of the invention,another method for detecting a replication competent HCV polynucleotideincludes measuring the production of viral particles by a cell. Themeasurement of viral particles can be accomplished by passage ofsupernatant from media containing a cell culture that may contain areplication competent HCV, and using the supernatant to infect a secondcell. Detection of HCV in the second cell indicates the initial cellcontains a replication competent HCV. The production of infectious virusparticles by a cell can also be measured using antibody thatspecifically binds to an HCV viral particle. As used herein, an antibodythat can “specifically bind” an HCV viral particle is an antibody thatinteracts only with the epitope of the antigen (e.g., the viral particleor a polypeptide that makes up the particle) that induced the synthesisof the antibody, or interacts with a structurally related epitope.“Epitope” refers to the site on an antigen to which specific B cellsand/or T cells respond so that antibody is produced. An epitope couldincludes about 3 amino acids in a spatial conformation which is uniqueto the epitope. Generally an epitope includes at least about 5 suchamino acids, and more usually, consists of at least about 8-10 suchamino acids. Antibodies to HCV viral particles can be produced asdescribed herein.

In another aspect, identifying a replication competent HCVpolynucleotide includes incubating a cultured cell that includes an HCVof the present invention. In those aspects of the invention where theheterologous polynucleotide encodes a detectable marker, cellscontaining a replication competent HCV can be identified by observingindividual cells that contain the detectable marker. Alternatively, ifthe detectable marker is secreted by the cell, the presence of themarker in the medium in which the cell is incubated can be detected.Methods for observing the presence or absence of a detectable marker ina cell or in liquid media are known to the art.

Another aspect of the invention provides for the positive selection ofcells that include a replication competent HCV polynucleotide. Themarker expressed by the HCV is a selectable marker, and the cell, whichincludes the HCV, is incubated in the presence of a selecting agent.Those cells that can replicate in the presence of the selecting agentcontain an HCV that is replication competent. Typically, the cells thatcan replicate are detected by allowing resistant cells to grow in thepresence of the selecting agent.

In some aspects, the method may further include isolating virusparticles from the cells that contain a replication competent HCVpolynucleotide and exposing a second cell to the isolated virus particleunder conditions such that the virus particle is introduced to the cell.After providing time for expression of the selectable marker, the secondcell is then incubated with the selecting agent. The presence of a cellthat replicates indicates the replication competent HCV producesinfectious virus particles. Preferably, virus particles are isolated byremoving a volume of the media in which the first cells are incubated.

In another aspect, the invention provides a method for detecting areplication competent HCV polynucleotide. The method includes incubatinga cell that contains an HCV of the present invention. The cell includesa transactivated coding region and an operator sequence operably linkedto the transactivated coding region. The transactivated coding regionencodes a detectable marker.

The heterologous polynucleotide present in the HCV polynucleotideencodes a transactivator that interacts with the operator sequencepresent in the cell. The interaction of the transactivator to theoperator sequence can decrease transcription or increase transcriptionof the operably linked transactivated coding region. Preferably, bindingof the transactivator to the operator sequence increases transcription.Preferably, the HCV also encodes a marker, more preferably, a fusionpolypeptide that includes a transactivator and a marker. Mostpreferably, the fusion polypeptide further includes a cis-actingproteinase located between the nucleotides encoding the transactivatorand the nucleotides encoding the marker.

The method further includes detecting the presence or absence of thedetectable marker encoded by the transactivated coding region present inthe cell. The presence of the detectable marker indicates the cellincludes a replication competent HCV. Preferably, the detectable markeris one that is secreted by the cell, for instance secretory alkalinephosphatase.

The methods described above for identifying replication competent HCVpolynucleotide can also be used for identifying a variant HCVpolynucleotide, i.e., an HCV that is derived from a replicationcompetent HCV of the present invention. Preferably, a variant HCV has afaster replication rate than the parent or input HCV. The method takesadvantage of the inherently high mutation rate of RNA replication. It isexpected that during continued culture of a replication competent HCV incultured cells, the HCV of the present invention may mutate, and somemutations will result in HCV with greater replication rates. The methodincludes identifying a cell that has greater expression of a polypeptideencoded by a replication competent HCV. An HCV of the present inventionthat replicates at a faster rate will result in more of thepolypeptide(s) that is encoded by the heterologous polynucleotidepresent in the HCV. For instance, when an HCV encodes a selectablemarker, a cell containing a variant HCV having a greater replicationrate will be resistant to higher levels of an appropriate selectingagent. When an HCV encodes a transactivator, a cell containing a variantHCV having a greater replication rate than the parent or input HCV willexpress higher amounts of the transactivated coding region that ispresent in the cell. The observed increases in resistance to phleomycinD1 (for instance, ZEOCIN) suggest the accumulation of mutations thatallow increased rates of replication.

A cDNA molecule of a variant HCV polynucleotide can be cloned usingmethods known to the art (see, for instance, Yanagi et al., Proc. Natl.Acad. Sci., USA, 94, 8738-8743 (1997)). The nucleotide sequence of thecloned cDNA can be determined using methods known to the art, andcompared with that of the input RNA. This allows identification ofmutations that have occurred in association with passage of the HCV incell culture. For example, using methods known to the art, includinglongrange RT-PCR, extended portions of a variant HCV genome can beobtained. Multiple clones could be obtained from each segment of thegenome, and the dominant sequence present in the culture determined.Mutations that are identified by this approach can then be reintroducedinto the background of the HCV cDNA encoding the parent or input HCV.This may be used to produce a replication competent HCV that does notcontain a heterologous polynucleotide. Such an HCV would have superiorreplication properties in cell culture compared to the parent HCV andthe variant HCV because it would not carry the burden of an additionalcoding region within its 3′ non-translated RNA.

The present invention also provides methods for identifying a compoundthat inhibits replication of an HCV polynucleotide, preferably areplication competent HCV as described herein in the section “HepatitisC Virus.” The method includes contacting a cell containing a replicationcompetent HCV polynucleotide with a compound and incubating the cellunder conditions that permit replication of the replication competentHCV polynucleotide in the absence of the compound. After a period oftime sufficient to allow replication of the HCV polynucleotide, thereplication competent HCV polynucleotide is detected. A decrease in thepresence of replication competent HCV polynucleotide in the cellcontacted with the compound relative to the presence of replicationcompetent HCV polynucleotide in a cell not contacted by the compoundindicates the compound inhibits replication of a replication competentHCV. A compound that inhibits replication of an HCV includes compoundsthat completely prevent replication, as well as compounds that decreasereplication. Preferably, a compound inhibits replication of areplication competent HCV by at least about 50%, more preferably atleast about 75%, most preferably at least about 95%.

The compounds added to a cell can be a wide range of molecules and isnot a limiting aspect of the invention. Compounds include, for instance,a polyketide, a non-ribosomal peptide, a polypeptide, a polynucleotide(for instance an antisense oligonucleotide or ribozyme), or otherorganic molecules. The sources for compounds to be screened include, forexample, chemical compound libraries, fermentation media ofStreptomycetes, other bacteria and fungi, and extracts of eukaryotic orprokaryotic cells. When the compound is added to the cell is also not alimiting aspect of the invention. For instance, the compound can beadded to a cell that contains a replication competent HCV.Alternatively, the compound can be added to a cell before or at the sametime that the replication competent HCV is introduced to the cell.

Typically, the ability of a compound to inhibit replication of areplication competent HCV polynucleotide is measured using methodsdescribed herein. For instance, methods that use nucleic acidamplification to detect the amount of HCV nucleic acid in a cell can beused. Alternatively, methods that detect or select for a marker encodedby a replication competent HCV or encoded by a cell containing areplication competent HCV can be used.

In some aspects of the invention, the replication competent HCVpolynucleotide of the invention can be used to produce infectious viralparticles. For instance, a cell that includes a replication competentHCV can be incubated under conditions that allow the HCV to replicate,and the infectious viral particles that are produced can be isolated,preferably purified. The infectious viral particles can be used as asource of virus particles for various assays, including evaluatingmethods for inactivating particles, excluding particles from serum,identifying a neutralizing compound, and as an antigen for use indetecting anti-HCV antibodies in an animal. An example of using a viralparticle as an antigen includes use as a positive-control in assays thattest for the presence of anti-HCV antibodies.

For instance, the activity of compounds that neutralize or inactivatethe particles can be evaluated by measuring the ability of the moleculeto prevent the particles from infecting cells growing in culture or incells in an animal. Inactivating compounds include detergents andsolvents that solubilize the envelope of a viral particle. Inactivatingcompounds are often used in the production of blood products andcell-free blood products. Examples of compounds that can be neutralizinginclude a polyketide, a non-ribosomal peptide, a polypeptide (forinstance, an antibody), a polynucleotide (for instance, an antisenseoligonucleotide or ribozyme), or other organic molecules. Preferably, aneutralizing compound is an antibody, including polyclonal andmonoclonal antibodies, as well as variations thereof including, forinstance, single chain antibodies and Fab fragments.

Viral particles produced by replication competent HCV polynucleotide ofthe invention can be used to produce antibodies. Laboratory methods forproducing polyclonal and monoclonal antibodies are known in the art(see, for instance, Harlow E. et al. Antibodies: A laboratory manualCold Spring Harbor Laboratory Press, Cold Spring Harbor (1988) andAusubel, R. M., ed. Current Protocols in Molecular Biology (1994)), andinclude, for instance, immunizing an animal with a virus particle.Antibodies produced using the viral particles of the invention can beused to detect the presence of viral particles in biological samples.For instance, the presence of viral particles in blood products andcell-free blood products can be determined using the antibodies.

The present invention further includes methods of treating an animalincluding administering neutralizing antibodies. The antibodies can beused to prevent infection (prophylactically) or to treat infection(therapeutically), and optionally can be used in conjunction with othermolecules used to prevent or treat infection. The neutralizingantibodies can be mixed with pharmaceutically acceptable excipients orcarriers. Suitable excipients include but are not limited to water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, neutralizing antibodies andpharmaceutically acceptable excipients or carriers may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvants which enhance the effectiveness ofthe neutralizing antibodies. Such additional formulations and modes ofadministration as are known in the art may also be used.

The virus particles produced by replication competent HCV polynucleotideof the invention can be used as a source of viral antigen to measure thepresence and amount of antibody present in an animal. Assays areavailable that measure the presence in an animal of antibody directed toHCV, and include, for instance, ELISA assays, and recombinant immunoblotassay. These types of assays can be used to detect whether an animal hasbeen exposed to HCV, and/or whether the animal may have an active HCVinfection. However, these assays do not use virus particles, but ratherindividual or multiple viral polypeptides expressed from recombinantcDNA that are not in the form of virus particles. Hence they are unableto detect potentially important antibodies directed against surfaceepitopes of the envelope polypeptides, nor are they measures offunctionally important viral neutralizing antibodies. Such antibodiescould only be detected with the use of infectious virus particles, suchas those that are produced in this system. The use of infectious viralparticles as antigen in assays that detect the presence of specificantibodies by virtue of their ability to block the infection of cellswith HCV viral particles, or that possibly bind to whole virus particlesin an ELISA assay or radioimmunoassay, will allow the detection offunctionally important viral neutralizing antibodies.

The present invention also provides a kit for identifying a compoundthat inhibits replication of a replication competent HCV polynucleotide.The kit includes a replication competent HCV polynucleotide as describedherein, and a cell that contains a polynucleotide including atransactivated coding sequence encoding a detectable marker and anoperator sequence operably linked to the transactivated coding sequencein a suitable packaging material. Optionally, other reagents such asbuffers and solutions needed to practice the invention are alsoincluded. Instructions for use of the packaged materials are alsotypically included.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material mayinclude a label which indicates that the replication competent HCVpolynucleoitde can be used for identifying a compound that inhibitsreplication of an HCV. In addition, the packaging material may containinstructions indicating how the materials within the kit are employed.As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic, and the like, capable of holding within fixedlimits the replication competent virus and the vertebrate cell.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Construction of the Infectious MK0-Z RNA

FIG. 1 shows the full-length modified HCV cDNA (MK0-Z) that wasconstructed by modification of pCV-H77C. The nucleotide sequence ofMK0-Z is shown in FIG. 9. A coding region encoding a polypeptideconferring resistance to neomycin has been expressed under control ofthe EMCV IRES from a second reading frame inserted within the 3′non-translated RNA in subgenomic Kunjin virus replicons. However, thespecific placement of the foreign sequence could not be used as a guidefor the placement of a coding region in HCV since the 3′ non-translatedRNA of these viruses share no sequence identity. In the case of MK0-Z,the heterologous sequence functions as a unique 3′ cistron, with theinternal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV)directing the cap independent translation of a novel polyproteincomposed of Tat and the ZEOCIN (phleomycin, Invitrogen) resistanceprotein, Zeo, separated by the cis-active 2A proteinase offoot-and-mouth disease (FMDV) virus. The Asn-Pro-Gly sequence at thecarboxy terminus of FMDV 2A mediates proteolytic cleavage at the 2AZeojunction, effectively separating the upstream Tat and downstream Zeopolypeptides (Ryan et al., EMBO J, 13, 928-933 (1994)). The heterologoussequence is placed within the 3′NTR of HCV, a genomic region thatcontains highly conserved sequences that cannot be deleted without lossof infectivity. More specifically, the heterologous sequence was placedwithin the variable region of the 3′NTR (FIG. 2). As a control, areplication-incompetent variant of MK0-Z, dS-MK0-Z, was constructed byopening the clone at two closely positioned Sma I sites within the NS5Bcoding region, then religating the plasmid. This resulted in aframe-shift deletion in the HCV sequence, upstream of the GDD motif inthe polymerase encoded by the NS5B coding region, that is lethal toviral replication. The novel 3′ reading frame in MK0-Z, has been shownto be active translationally in in vitro translation reactions carriedout in rabbit reticulocyte lysates. These experiments also demonstratedthat the 2A proteinase effectively cleaved the resulting polyprotein,releasing Tat-2A from the Zeo protein.

a. Construction of pUC HCV3′-EMCV-tat-2A-Zeo

To make pHCV3′, full length HCV 1a (present on the plasmid pCV-H77C)(provided by Dr. Purcell at NIH) was digested with HindIII-XbaI. A DNAfragment of about 1.7 kilobases, corresponding to nucleotides 7861-9599of the HCV nucleotide sequence available at Genbank Accession numberAF011751, was isolated and ligated into the vector pBluescript(Stratagene) that had been digested with HindIII and XbaI. The resultingplasmid was designated pHCV3′.

A DNA fragment containing the EMCV IRES was generated by the polymerasechain reaction (PCR). The plasmid pEMCV-CAT, described in Whetter etal., (Arch. Virol. Suppl. 9, 291-298 (1994)) was amplified using thesense primer 5′-GGCCTCTTAAGGTTATTTTCCACCATATTGCC (SEQ ID NO:22) whichcontained a BfrI site, and the anti-sense primer5′-TCCCCGCGGAAGGCCTCATATTATCATCGTGTTTTTC (SEQ ID NO:23) which containeda SacI and StuI site. The italicized nucleotides are those which are notpresent in the DNA to be amplified, and the underlined nucleotidesindicate a restriction endonuclease site. The PCR conditions were: 94°C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute, for35 cycles.

pHCV3′-EMCV was generated by ligating EMCV IRES fragment digested withBfrI-SacI and vector from pHCV3′ digested with same enzymes.

A DNA fragment containing the nucleotides encoding 85 amino acids fromthe HIV I Tat protein was generated by PCR. The amino acid sequence ofthe HIV I Tat protein is shown at amino acids 4-89 of SEQ ID NO:21 Theplasmid used was pCTAT (provided by Dr. Bryan Cullen, Duke University,Durham, N.C. Dept. of Microbiology) (see Bieniasz et al., MolecularCellular Biology, 19, 4592-4599); was amplified using the sense primer5′-GAAGGCCTATGGAGCCAGTAGATCCTAGA (SEQ ID NO:28), which contained a StuIsite, and and anti-sense primer 5′-CGGAA TTCTTCCTTCGGGCCTGTCGGGTCC (SEQID NO:29), which contained an EcoRI site. The italicized nucleotides arethose which are not present in the DNA to be amplified, and theunderlined nucleotides indicate a restriction endonuclease site. The PCRconditions were: 94° C. for 30 seconds, 55° C. for 30 seconds, and 72°C. for 1 minute, for 35 cycles.

A DNA fragment containing the nucleotides encoding 15 amino acids ofFMDV 2A was generated by annealing 51 mer primer set; sense primer 5′-AATTCGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCTGGGCCC G (SEQ ID NO:24) andanti-sense primer 5′-GA TCCGGGCCCAGGGTTGGACTCGACGTCTCCCGCAAGCTTAAGAAGGTCG (SEQ ID NO:25) with putative digested form of EcoRI and BamHI site atits 5′ and 3′ end, respectively. The result was a DNA fragment encodingthe 15 amino acids of FMDV 2A. The amino acid sequence encoded by theDNA fragment was FDLLKLAGDVESNPG (SEQ ID NO:30).

A DNA fragment containing the coding region encoding resistance tophleomycin was generated by the polymerase chain reaction (PCR). Theplasmid pZeoSV (Invitrogen) was amplified using the sense primer5′-CCGCTCGAGGCCTGGATCCATGGCCAAGTTGACCAGTGCC (SEQ ID NO:26) whichcontained a BamHI site, and anti-sense primer5′-GGCCTCTTAAGTCAGTCCTGCTCCTCGGCCACG (SEQ ID NO:27) which contained aBfrI site. The italicized nucleotides are those which are not present inthe DNA to be amplified, and the underlined nucleotides indicate arestriction endonuclease site. The PCR conditions were: 94° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 1 minute, for 35 cycles.

pΔHCV3′-2A-Zeo was generated by digesting the DNA fragment containingthe coding region encoding resistance to phleomycin with BfrI-BamHI, andpHCV3′ was with EcoRI-BfrI. These two fragments and the FMDV 2A fragment(which contains an EcoRI site with staggered ends and a BamH site withstaggered ends) were then ligated to form pΔHCV3′-2A-Zeo.

pUC HCV3′-EMCV-tat-2A-Zeo was generated by ligating 4 fragmentstogether. A DNA fragment containing the EMCV IRES was obtained bydigesting pHCV3′-EMCV with SphI-StuI. The amplified DNA fragmentencoding a portion of the HIV I Tat protein was digested withStuI-EcoRI. pΔHCV3′-2A-Zeo was digested with EcoRI and XbaI to yield aDNA fragment containing the nucleotides encoding the FMVD 2A andphleomycin resistance. pUC20 vector digested with SphI-XbaI. These wereligated together and the resulting plasmid was designated pUCHCV3′-EMCV-tat-2A-Zeo.

b. Construction of pUC HCV3′-EMCV-tat-2A Containing New HCV 3′Fragment

Original full length HCV 1a (present on the plasmid pCV-H77C) wasdigested with SphI-BfrI and a 342 nucleotide fragment (corresponding tonucleotides 9060-9427 of HCV) was isolated. pUC HCV3′-EMCV-tat-2A-Zeowas digested StuI-BamHI and a fragment of 317 nucleotides containingtat-2A was isolated. The remaining portion of the plasmid was digestedwith BfrI, and a 508 nucleotide BfrI-StuI fragment containing the EMCVIRES was isolated. The remaining 361 nucleotide fragment, whichcontained the nucleotides encoding phleomycin resistance was isolatedand reserved for later use in the construction of pUC Zeo-HCV3′NTRcontaining new HCV3′NTR fragment (see section c below).

pUC HCV3′-EMCV-tat-2A was generated by ligating the 3 fragmentsdescribed above, i.e., the 342 nucleotide SphI-BfrI fragmentcorresponding to nucleotides 9060-9427 of HCV, the 508 nucleotideBfrI-StuI fragment containing the EMCV IRES, and the 317 nucleotideStuI-BamHII fragment containing tat-2A, with the vector pUC20 that hadbeen digested with SphI-BamHI. The resulting plasmid was designated pUCHCV3′-EMCV-tat-2A.

c. Construction of pUC Zeo-HCV3′NTR Containing New HCV3′NTR Fragment

pUC Zeo-HCV3′NTR was constructed by ligating the 361 nucleotideBamHI-BfrI fragment encoding phleomycin resistance (see above), a 198nucleotide fragment (corresponding to nucleotides 9427-9625 of HCV)generated by digesting original full length HCV 1a with BfrI-XbaI, andthe vector pUC20 that had been digested with BamHI-XbaI.

d. Construction of MK0-Z RNA

Steps b and c above were repeated to produce a second pUCHCV3′-EMCV-tat-2A and a second pUC Zeo-HCV3′NTR containing new HCV3′NTRfragment for use in the construction of MK0-Z RNA.

MK0-Z was generated by the ligation of 4 fragments. Full length HCV wasdigested with HindIII-SphI and a 1,199 nucleotide fragment(corresponding to nucleotides 7861-9060 of HCV) was isolated. ASphI-BamHI DNA fragment containing HCV3′-EMCV-tat-2A was isolated frompUC HCV3′-EMCV-tat-2A. A BamHI-XbaI DNA fragment containing Zeo-HCV3′NTRwas isolated from pUC Zeo-HCV3′NTR. Nucleotides corresponding tonucleotides 1-7860 were isolated from pCV-H77C by digestion withHindIII-XbaI. Ligation of these 4 fragments resulted in MK0-Z.

e. Construction of ds-MK0-Z RNA

The plasmid pHCV3′ was digested with SmaI and ligated under conditionsto result in self-ligation. The result of the self ligation was loss ofthe nucleotides corresponding to nucleotides 8497-8649 of HCV. Theresulting plasmid was designated pds-HCV3′.

ds-MK0-Z was generated by ligation of 4 DNA fragments. pds-HCV3′ wasdigested with HindIII-SphI to yield a DNA fragment corresponding tonucleotides 7861-9060 of HCV and containing the SmaI fragment deletion.pUC HCV3′-EMCV-tat-2A was digested with SphI-BamHI to yield a fragmentcontaining HCV3′-EMCV-tat-2A. pUC Zeo-HCV3′NTR was digested withBamHI-XbaI to yield a fragment containing the nucleotides encodingZeo-HCV3′NTR. Nucleotides corresponding to nucleotides 1-7860 wereisolated from pCV-H77C by digestion with HindIII-XbaI. Ligation of these4 fragments resulted in ds-MK0-Z.

Example 2 Production of the Virus by Chimpanzee

This demonstrates the insertion of a heterologous sequence into an HCVdoes not destroy the ability of the HCV to replicate and produceinfectious virus.

MK0-Z plasmid was linearized with XbaI and RNA was synthesized with T7mega transcription kit from Ambion. The reaction was analysed by gelelectrophoresis before injecting into the liver of an HCV-naiveChimpanzee. RNA was frozen at −70° C. overnight before used. About 300μg of RNA was injected. When injecting, the RNA, which was in 100 ml oftranscription reaction mixture, was diluted in 1 ml PBS. The RNA wasadministered to a Chimpanzee by percutaneous intrahepatic injectionguided by ultrasound. Several sites and injections were done in singleday. The levels of ALT in the chimpanzee were monitored and were innormal ranges throughout the experiment. Sera from the chimpanzee werecollected weekly, and the presence of HCV in each 1 ml of those sera,were checked by RT-PCR, using either the TaqMan or Light Cycler RT-PCRmethods.

The primers and probe used for the TaqMan RT-PCR were sense primer,AAGACTGCTAGCCGAGTAGTGTT nt 243 to 265 (SEQ ID NO:1); anti-sense primer:GGTTGGTGTTACGTTTGGTTT nt 390 to 370 (SEQ ID NO:2); and probe:TGCACCATGAGCACGAATCCTAAA nt 336 to 359 (SEQ ID NO:3), where “nt 243 to265,” “nt 390 to 370,” and “nt 336 to 359” refers to the HCV nucleotides(at Genbank Accession number AF011751) to which the primers hybridize.All single-tube EZ RT-PCR reactions were carried out in optical MicroAmpreaction tubes with optical lids in 50 microliter (μl) volume (96 wellformat). The RNA amplification was done using the TaqMan EZ RT-PCR Kit.Briefly, reactions contained 1× amplification buffer (TaqMan EZ Buffer),3 mM manganese, 0.5 U AmpErase uracil-N-glycosylate, 7.5 U rTth DNApolymerase, RNA, 200 nM forward and reverse primers, 200 μM each dNTP,and 500 uM of dUTP. Thermocycling conditions were one cycle at 50° C.for 2 minutes, one cycle at 60° C. for 30 minutes, one cycle at 95° C.for 5 minutes, and 40 cycles of 95° C. for 20 seconds, 60° C. for 1minute. Amplifications were evaluated by ABI7700 Sequence Detectorversion 1.6.3 software (Applied Biosystems), as suggested by themanufacturer.

The primers and probe used for Light Cycler RT-PCR were forward primer,ACACTCCACCATGAATCACTC, nt 22 to 41, (SEQ ID NO:4); reverse primer,GATCGGGCTCATCACAACCC, nt 268 to 250, (SEQ ID NO:5); fluor probe,GCGTCTAGCCATGGCGTTAGTATGAGT(fluor), nt 75 to 101 (SEQ ID NO:6); and redprobe, (LC640) TCGTGCAGCCTCCAGGACCCC(phosphate), nt 103 to 123 (SEQ IDNO:7). The terms “nt 22 to 41,” “nt 268 to 250,” “nt 75 to 101” and “nt103 to 123” refer to the HCV nucleotides (at Genbank Accession numberAF011751) to which the primers hybridize. The “fluor probe” is labeledat the 3′ end with fluorescein, and the “red probe” is labeled at the 5′with LightCycler Red 640 dye.

Single-tube RT-PCR reactions were carried out in capillary tubes in areaction volume of 20 μl using the core reagents of RNA AmplificationKit Hybridization Probes (Roche) as suggested by the manufacturer. Amaster mix was made according to the manufacturer's suggestions,containing Lightcycler-RT-PCR Reaction Mix Hybridization probe solution,LightCycler RT-PCR Enzyme mix, 7 mM MgCl₂, 0.5 μM of forward primer, 0.9μM of reverse primer and 0.5 μM of fluor probe, 0.9 μM of red probe, andH₂O is added to make it total 20 μL. This master mix was added directlyto the RNA pellet and after dissolve the RNA, it was loaded into glasscapillary tube. After adding the 5 ul wash, the tube was snap sealedwith a plastic cap. The RT-PCR conditions were 55° C. for 15 minutes,95° C. for 30 seconds, and 40 cycles of 94° C. for 0 seconds, 60° C.annealing for 15 seconds, and 72° C. extension for 15 seconds.

The signal acquisition was at the end of the annealing step for 100milliseconds (ms). After amplification was complete, a melting curve wasperformed by cooling to 55° C., holding at 55° C. for 30 seconds, andthen heating slowly at the rate of 0.2 C/second until 90° C. Signal wascollected continuously during this melting to monitor the dissociationof the 5′-LC640-labeled probe. The signal was the result of fluorescenceresonance energy transfer (FRET) between the fluor probe and the redprobe. These probes hybridize to an internal sequence of the amplifiedfragment during the annealing phase of the PCR cycle. One probe islabeled at the 5′ end with a LightCycler-Red fluorophore (LC-Red 640 orLC-Red 705), and to avoid extension, modified at the 3′ end byphosphorylation. The other probe is labeled at the 3′ end withfluorescein. Only after hybridization to the template, do the two probescome in close proximity, resulting in FRET between the two fluorophores.During FRET, fluorescein, the donor fluorophore, is excited by the lightsource of the LightCycler Instrument. Part of the excitation energy istransferred to LightCycler-Red, the acceptor fluorophore. The emittedfluorescence of the LightCycler-Red fluorophore is measured. The meltingcurves were then displayed as −dF/d T vs T plots as calculated byLightCycler software version 3.

The results of TaqMan RT-PCR are shown in FIG. 11. They demonstrate thatMK0-Z RNA is infectious in a chimpanzee.

Example 3 Construction of a Cellular Enzyme Reporter System forDetection of Replicating HCV

A major difficulty in evaluating the outcome of experiments in whichcultured cells are transfected with candidate infectious RNAs lies inthe detection of newly synthesized viral RNAs against the largebackground of transfected input RNA. While this is less of a problemwith very robustly replicating viral RNAs, only Lohmann et al. (Science,285,110-113 (1999)) and Blight et al. (Science, 290, 1972-1975 (2000))have thus far reported levels of replication detectable by northernanalysis, using subgenomic RNA replicons that are not capable ofproducing infectious virus. Moreover, these authors observed suchreplication only in a small number of cell clones that were isolatedover a period of weeks by a stringent antibiotic selection protocol.RT-PCR is difficult to use to detect newly replicated nucleic acid inrecently transfected cells due to the persistence of input RNA (in ourexperience, RNA transfected by liposome-mediated methods remainsdetectable for weeks). The use of a negative-strand “specific” assayreduces, but does not eliminate this problem, since such assays have nomore than a −1,000-fold relative specificity for detection of thenegative strand vs. detection of the positive-strand (see, for instance,Lanford et al., J. Virol., 69, 8079-8083 (1995)).

This Example details the construction of a cell line that allows thedetection of replicating synthetic HCV RNA. The detection is based onthe detection of a protein product expressed from the RNA. The systemuses the incorporation of the sequence encoding the HIV I Tat proteinwithin modified viral RNAs (see FIG. 1). The Tat protein is a strongtransactivator of the HIV I long terminal repeat (LTR) transcriptionalregulator. For use as cell substrates in this system, multiple stablytransformed cell lines were established. The transformed cell lines werederived from Huh-7 cells that express secretory alkaline phosphatase(SEAP) under transcriptional control of the HIV I LTR. These cell lineswere established using either Neomycin or Blastocidin selection, so thateither of these antibiotics or Zeocin can be used for subsequentselection of replicating full-length HCV RNAs. The expression of Tatwithin these cells leads to measurable increases in SEAP activity withinthe culture medium, as depicted schematically in FIG. 3.

For establishment of neomycin resistant SEAP cell lines, the HIV-SEAPsequence was PCR amplified from pBCHIVSEAP plasmid (provided by Dr.Bryan Cullen, Duke University, Durham, N.C. Dept. of Microbiology) (seeCullen, Cell, 46, 973-982 (1986), and Berger et al., Gene, 66, 1-10(1988)) using the primer pairs 5′-CTAGCTAGCCTCGAGACCTGGAAAAACATGGAG (SEQID NO:8) and 5′-ATAAGAATGCGGCCGCTTAACCCGGGTGCGCGG (SEQ ID NO:9). Thenon-italicized nucleotides in SEQ ID NOs:8 and 9 hybridize withnucleotides present in the target DNA, and the italicized nucleotides inSEQ ID NO:9 represent additional nucleotides that do not hybridize withthe target DNA. The underlined nucleotides indicate introducedrestriction endonuclease sites. The nucleotide sequence of the amplifiedfragment is shown in FIG. 12 (SEQ ID NO:18).

After filling in to repair the possible PCR overhang, this fragment wasdigested with NotI and ligated to vector derived from pRcCMV(Invitrogen) digested with NruI-NotI removing CMV promoter. Theresulting plasmid was designated pRcHIVSEAP The nucleotide sequence ofthe pRcHIVSEAP was used to transfect Huh-7 cells using a non-liposomaltransfection reagent commercially available under the trade name FUGENE(Boerhinger Manheim). Tranfectants were selected using G418 (neomycin).The ability of a cell to express SEAP in the presence of tat was testedby transfecting cells with the plasmid pCTAT, which expresses the tatprotein. Two resulting cell lines which expressed high levels of SEAPwere designated Huh-o10 (also referred to as Huh7-SEAP-o10) andHuh7-SEAP-N7, and were used for subsequent experiments.

A Blasticidin resistant SEAP cell line was constructed as follows.pcDNA6/V5-His (Invitrogen) was digested with BglII-BamHI to remove theCMV promoter. The vector was then self-ligated and subsequently digestedwith EcoRV-NotI and ligated to the HIV-SEAP DNA fragment that was PCRamplified from pBCHIVSEAP fragment mentioned. The resulting plasmid wasused to transfect Huh-7 cells using a non-liposomal transfection reagentcommercially available under the trade name FUGENE (Boerhinger Manheim).Tranfectants were selected using Blastocidin (Invitrogen). A blastocidinresistant cell was selected and designated Huh-SEAP-Bla-EN.

Example 4 Evaluation of the Cellular Enzyme Reporter System forDetection of Replicating HCV

This Example demonstrates the feasibility and utility of the SEAPcellular reporter system, and demonstrates the expression of Tat by thegenetically modified HCV RNA.

To test the SEAP cellular reporter system, MK0-Z RNA was synthesized andtransfected into two different SEAP reporter cell lines, Huh7-SEAP-o 10and Huh7-SEAP-N7 (another cell line that resulted from neomycinselection), on the same day. To provide adequate controls for thisexperiment, cells from both cell lines were transfected with RNAssynthesized from each of the plasmid DNAs shown in FIG. 1. These includeMK0-Z, its replication incompetent control dS-MK0-Z, and a subgenomictranscript, 3′ETZ, each of which encode the novel polyprotein consistingof Tat and Zeo separated by the 19 amino acid 2A proteinase from FMDV 4.Fifteen of the amino acids were the FMDV 2A sequence, and 4 additionalamino acids were encoded by nucleotides present to introduce restrictionendonuclease sites. In each of the transfected RNAs, this polyprotein isunder the translational control of the EMCV IRES.

DNA was linearized with Xba I and RNA was synthesized with T7 megatranscription kit (Ambion, Madison, Wis.). Transfection of RNA was doneusing Lipofectin (Gibco BRL, Rockville, Md.). Briefly, about 5 μg of RNAwas added to a mixture (1 hour incubation prior to transfection) of 15μl of Lipofectin and 200 μl OPTIMEM (Gibco BRL), incubated for 15 min,and applied to cells. The cells were in 6 well plates which had beenplated one day before transfection. The cells were washed two times withOPTIMEM before addition of the RNA, followed by the addition of 1 ml ofOPTIMEM. After overnight incubation, cells were washed with PBS twotimes and growth medium (DMEM with 2% FBS as above) was added.

Transfection of these RNAs was associated with striking increases inSEAP secreted into the cell culture supernatant, as measured by assay ofSEAP. SEAP was assayed using Tropix Phospha-Light ChemiluminescentReporter Assay for secreted Alkine Phosphatase reagent (Tropix, FosterCity, Califonia), according to the manufacturer's suggested protocol,but reduced 1/3 in scale. Luminescent signal detected by a TD-20/20Luminometer (Turner Design).

The increase in SEAP occurred as a result of transfection with eitherMK0-Z or the replication deficient dS-MK0-Z RNA, indicating that theSEAP released in the initial weeks after transfection was expressed fromthe input RNA, not newly replicated RNA. High expression of SEAP wasobserved from 3′ETZ, reflecting greater transfection efficiency of thissmall RNA transcript. This experiment demonstrates the feasibility andutility of the SEAP cellular reporter system, and demonstrates theexpression of Tat by the genetically modified HCV RNA.

Proof that infection had been accomplished by the transfection of MK0-ZRNA and that virus adaptation to replication in cultured cells hadoccurred under antibiotic selection pressure accumulated over theensuring several months, as follows. FIG. 4 (left panel) shows theresults of SEAP assays on media harvested from these cells during thefirst month after transfection with MK0-Z, and the pol(−) mutantdSMk0-Z. These cells were subsequently maintained in medium with a lowconcentration of fetal calf serum (2%) over the ensuing 3 months, duringwhich the cells were split periodically and intermittently exposed tolow concentrations of the antibiotic Zeocin as tolerated (about 10 to 25μg/ml). There was no significant difference in cell survival in thepresence of Zeo between cells transfected with MK0-Z, and thosetransfected with dSMK0-Z, but the former usually expressed somewhathigher levels of SEAP in the media (about 1.5 times to about 2 timeshigher than the control cells). At approximately 3 months, these cells(both MK0Z and ds-MKM0-Z transfected cells) underwent a spontaneouscrisis with loss of viability. The supernatant fluids were collected andplaced on replicate cultures of fresh Huh-SEAPo 10 cells in an attemptat blind passage of virus. Antibiotic selection was continuedintermittently, with gradually intensifying Zeocin selection(intermittent exposure ultimately to 50 μg/ml). With the increase to 50μg/ml Zeocin, sudden marked increases in SEAP expression were noted fromreplicate cultures of cells that had been inoculated with medium fromthe MK0-Z transfected cells, but not cells inoculated with the pol(−)mutant, dS-MK0-Z. This occurred about 7 months after the originaltransfection, and 4 months after the attempt at cell-free passage ofvirus. All cells were unable to survive the higher concentration of Zeo,however and the cultures were lost at this point. However, cells thathad been previously frozen from the putative passage were recovered fromthe freezer, and subjected to intermittent concentrations of Zeocinranging from 25-50 μg/ml. Results are shown in FIG. 5, and summarized inTable 2. TABLE 2 Passage history of vMK0-Z —infected Huh-SEAP-o10 C-Aand C-B sublines.¹ Approximate elapsed Passage time (days) Comments P1 1Huh-SEAP-o10 cells transfected with MK0-Z RNA, maintained in the absenceof antibiotic selection. 33 Start intermittent Zeocin selectionpressure, 75 10-25 mg/ml. Cells entered crisis and were lost P2 68 FreshHuh-SEAP-o10 cells infected with P1 day 68 supernatant, and maintainedin intermittent Zeocin 25 mg/ml. 190 Increase Zeocin to 25-50 mg/ml,with resulting increase in SEAP expression. 197 Cells frozen(continuously cultured cells lost within about 1.5 months) 283 Cellsfrozen on P2 day 197 were replated, cultured in intermittent Zeocin50-100 mg/ml, with marked increase in SEAP expression. P2 cells infectedwith P1 supernatant from control dS-MK0-Z did not survive. 547 Two celllines (C-A and C-B), both established on P2 day 283, maintained inintermittent Zeocin 50-100 mg/ml with high SEAP. P3 514 FreshHuh-SEAP-o10 cells infected with 0.45 m-filtered supernatant media fromP2 C-A and C-B cell lines on day 544, maintained in intermittent Zeocin25 mg/ml.¹The term “vMK0-Z” is used to refer to the viral form of MK0-Z afterpassage.

As observed previously, striking increases occurred in the level of SEAPsecreted from 12 of 12 replicate cultures of cells infected with mediumfrom the MK0-Z-transfected cells, but not from any cultures of cellsinfected in parallel with medium from dS-MK0-Z transfected cells.Moreover, all of the control cell cultures were lost under exposure to50 μg/ml Zeocin, while each of the cultures infected with MK0-Z materialremained viable. Significantly, there was no increase in SEAP releasedinto the medium from the dying cell lines (FIG. 5, dSma (C-A) and dSma(C-B)), consistent with the fact that all SEAP produced is activelysecreted from the cells into the medium. This result confirms that celldeath does not result in a false elevation of SEAP activity in culturesupernatant fluids. The Zeocin resistance and SEAP expression displayedby these cells cannot be explained by fortuitous integration of DNA fromthe transfected material, since the cells shown in FIG. 5 were nevertransfected, only exposed to medium from transfected cells. Cellsurvival and SEAP expression also cannot be explained by cellularmutations in these experiments, as these events have occurred inmultiple cultures exposed to the supernatant fluid of MK0-Z transfectedcells, but not in related control cell cultures that were similarlyexposed to media from dS-MK0-Z transfected cells.

Fluctuations in SEAP activity correlated in part with cell density, andcell viability. At times, these cultures demonstrated considerablecytopathology. However, it was demonstrated that there was minimalintracellular SEAP activity and that most SEAP is actively secreted fromthe cells. Thus, peaks of SEAP activity reflect peaks of SEAP synthesis,not release from dying cells.

The results shown in FIG. 5 indicate that these cells express twoheterologous proteins encoded by MK0-Z, RNA. The Huh-SEAP-o 10 cellshave acquired relative Zeocin resistance, indicating the expression ofthe Zeocin resistance protein, and they secrete 5- to 10-fold greaterquantities of SEAP than control cells, indicating the expression of Tat.Moreover, RT-PCR has been used to successfully detect the presence ofHCV RNA in samples of the supernatant fluids collected from these cells,using a primer set derived from the viral 5′NTR (see Example 5).Detection of the signal was dependent on Southern blotting of firstround RT-PCR products, and amplification was dependent upon theinclusion of reverse transcriptase in the reaction. The results suggestthat only small quantities of RNA are present, but confirm that theRT-PCR products are amplified from RNA and not contaminating DNA. Thesequence of the amplified product was identical to the H77C strain 5NTR,the virus from which the MK0-Z clone was derived. These results thusrepresent the first successful attempt at recovery of HCV from cellstransfected with synthetic RNA.

One of the more important features of the experiment depicted in FIG. 5is the significant change in the behavior of these HCV infected cellsover the months of observation, both in terms of their increasing Zeocinresistance and increasing SEAP secretion. This is consistent withadaptation of the viral RNA to more efficient replication within thesecells, as would be expected for a positive-strand RNA virus.Furthermore, since at this point all of the cells exposed to medium fromcells transfected with the pol(−) mutant dS-MK0-7 have failed to surviveZeocin selection, it can now be assumed that all of the surviving cellsharbor viral RNA. Thus, any further increases in SEAP expression must beindicative of greater abundance of the RNA and enhanced replication ofthe virus.

In summary, these two cell lines continue to demonstrate substantialZeocin resistance and high level SEAP activity, two independent measuresof protein expression from the second open reading frame of the modifiedvMK0-Z genome, more than 12 months after their infection withsupernatant fluids taken from RNA-transfected cells. This is strongevidence of continued replication of the viral RNA in these cells.

Example 5 Passage of vMK0-Z to Fresh Huh-SEAP-o10 Cells

A third passage of vMK0-Z was carried out using supernatant mediacollected from the C-A and C-B cell lines on P2 day 540 (see Table 2).These media samples were passed through a 0.45μ filter and then used tofeed fresh Huh-SEAP-o10 cells. Control cell cultures (n=6) were mockinfected with normal media. One hundred and twenty hours afterinoculation, these cells were exposed to intermittent Zeocin selectionpressure (25 μg/ml). When treated with high concentrations of drug, orwhen maintained in continuous drug condition, these cells tend to die.Accordingly, drug exposure was intermittent, and not at highconcentrations. The mock-infected cells were lost due to Zeocin toxicityby about day 546 (relative SEAP activity of infected to control cells atthis point was 42658 and 31510, respectively, and is not shown in FIG.6).

The results shown in FIG. 6 demonstrate the passage of SEAP expressionactivity and Zeocin resistance to fresh Huh-SEAP-o10 cells followinginoculation of these cells with supernatant medium collected fromvMK0-Z-infected cells.

Example 6 Detection of Viral RNA in Huh-SEAP-o 10 Cell Lines

Despite the results described above, and the demonstration of viralantigen in MK0-Z infected cells (see Example 7), it has proven difficultto consistently demonstrate viral RNA in these cells. This Exampledescribes methods for detecting the presence of viral RNA inHuh-SEAP-o10 cell lines.

Two different quantitative RT-PCR assays (LightCycler and TaqMan) havebeen used in recent efforts to detect viral RNA in lysates of the cellsor in supernatant media. Greatest consistency of success has been indetection of viral RNA in supernatant media following PEG precipitation.This technique works very well, allowing concentration of 130 genomecopies equivalent from 1 milliliter (ml) supernatant with 80% recovery.Viral RNA has been reproducibly but intermittently detected in thesupernatant fluids; however, reliable detection of viral RNA in celllysates has not been possible.

The primers and probes that have been used for these assays were asfollows:

LighiCycler RT-PCR

This method used the Lightcycler thermal cycler manufactured by Roche.

Primers: Forward 5′-GACACTCCACCATGAATCACT, (SEQ ID NO: 10) nt 21 to 41,Reverse 5′-GTTCCGCAGACCACTATGG, (SEQ ID NO: 11) nt 156 to 139,

Probesforfluorescence Resonance Energy Transfer (FRET):5′-AGAAAGCGTCTAGCCATGGCGTTAG (SEQ ID NO: 12) (Fluor)5′(LC640)ATGAGTGTCGTGCAGCCTCCAG (SEQ ID NO: 13) (phosphate)

Briefly, the HCV virus was precipitated with PEG (Sigma, St. Louis, Mo.)prior to extraction with QIAamp serum kit Qiagen, Valencia, Calif.).Supernatant (1.3 ml) was mixed with 0.3 ml of 40% PEG and was placed inan ice bath for 4 hours. The mixture was then centrifuged at 10000×g for30 minutes at 4° C. The supernatant was removed from the white pelletand 140 μl of TE was added to it. The RNA was then extracted from theviral pellet by following the manufacturers instructions. The eluate wastreated with Dnase I as was instructed by the T7 mega transcription kit(Ambion), precipitated with 60 μg glycogen in 130 μl IPA, and stored at−80° C. The positive serum control was a volume of serum containing 5000genome equivalents, added to media (1.3 ml TE) before precipitation with0.3 ml PEG and extraction as discussed above. The HCV genome equivalentswere determined by National Genetics Institute (Los Angeles, Calif.).The negative serum control was 1 μl of serum from an uninfectedvolunteer. The serum was treated in the same way as the positive controlserum.

The single-tube RT-PCR reactions were carried out in capillary tubes ina reaction volume of 20 μl using the core reagents of RNA AmplificationKit Hybridization Probes (Roche). A 20 μl RT-PCR mixture contained 0.05μM forward primer, 0.9 μM of reverse primer, RNA sample and 5 ul tubewash of purified sample RNA. The precipitated RNA was firstreconstituted with RT-PCR master mix then was loaded into a glasscapillary tube, after adding the 5 μl wash the tube was snap sealed witha plastic cap. The RT-PCR conditions were 55° C. for 15 minutes, 95° C.for 30 seconds, and 40 cycles of 94° C. for 0 seconds, 60° C. annealingfor 15 seconds, and 72° C. extension for 15 seconds. The signalacquisition was at the end of the annealing step for 100 ms. Afteramplification was complete, a melting curve was performed by cooling to55°, holding at 55° C. for 30 seconds, and then heating slowly at 0.2C/seconds until 90° C. Signal was collected continuously during thismelting to monitor the dissociation of the 5′-LC640-labeled probe. Themelting curves were then displayed as −dF/d T vs T plots by LightCylersoftware version 3.

Results obtained in the LightCycler assay with PEG-precipitatedsupernatant media collected from the C-A and C-B cell sublines are shownin FIG. 7, which shows the melting curve detected by the FRET method.The melting curve indicates the specificity of product. Both C-A andC-B's curve matches that of positive control. The height of the curvecorrelates with the amount of the product produced. The negative mediacontrol was cell culture media maintained in the isolation room in whichthe C-A and C-B cell sublines are maintained. The negative serum controlwas contributed by a volunteer.

TaqMan RT-PCR

Primers (see Takeuchi et al., Gastroenterol., 116, 636-642 (1999)):Forward 5′-CGGGAGAGCCATAGTGG (SEQ ID NO: 14) Reverse5′-AGTACCACAAGGCCTTTCG (SEQ ID NO: 15)

TaqMan Probe: 5′-(FAM)-CTGCGGAACCGGTGAGTACAC (SEQ ID NO: 16) (TAMRA)-3′

RNA was obtained from cells as described above for PCR with theLightcycler thermal cycler. This experiment was set up according to theprotocol provided in TaqMan EZ RT-PCR Core Reagents Protocol (productnumber 402877, Applied Biosystems, Foster City, Calif.). Briefly, Allsingle-tube EZ RT-PCR reactions were carried out in optical MicroAmpreaction tubes with optical lids and in 50 μl volume in a 96-wellformat. The RNA amplification contained Ix amplification buffer, 3 mMmanganese, 0.5 Units (U) AmpErase uracil-N-glycosylate, 7.5 U rTth DNApolymerase, RNA, 200 nM forward and reverse primers, 200 μM each dNTP,500 μM of d UTP. AB17700 Sequence Detector version 1.6.3 software wasused for sample analysis. Thermocycling conditions were one cycle at 50°C. for 2 minutes, one cycle at 60° C. for 30 minutes, one cycle at 95°C. for 5 minutes, 40 cycles at 95° C. for 20 seconds and 60° C. for 1minutes.

FIG. 8 shows results of TaqMan RT-PCR The C-A and C-B product asdetected according to program is aligned along with a knownconcentration of positive control HCV. The approximate number of HCVprotracted from this graph is shown in Table 3. TABLE 3 TaqManquantitation of HCV RNA in supernatant media. Supernatant from: Numberof genome equivalents Positive serum control (5000 ge¹) 4188 C-B  109C-A  136 C-B (unhealthy culture)²   3 C-A (unhealthy culture)²   7Negative control media   24³ Medium   0 Negative control   0¹ge, genome equivalents.²Cultures were losing viability.³This is believed to be the result of contamination.

There was good correlation between the TaqMan and LightCycler results onthese specimens.

Example 7 Demonstration of Viral Antigens in vMK0-Z-InfectedHuh-SEAP-o10 Cell Lines

Viral antigens expressed from both coding regions (i.e., the codingregion encoding the viral polypeptides and the coding region inserted inthe 3′ NTR) in the modified HCV genome have been demonstrated in vMK0-Zinfected Huh-SEAP-o10 cells by indirect immunofluorescence. Negativecontrols for these experiments were uninfected Huh-SEAP-o10 cells. Cellswere grown in tissue culture chamber slides and fixed inacetone-methanol at room temperature prior to staining. Cells were fixedin 50% methanol/50% Acetone for 10 minutes. Blocking agent was 3% BSA inPBS. The primary antibodies used were a mouse monoclonal antibodyagainst HCV core protein, (anti-core antibody, provided by Johnson Lau,Schering-Plough Research Institute, Kennilworth, N.J.) used at adilution of 1:100, a rabbit polyclonal antibody raised against Sh Bleprotein (anti-Zeo antibody, CAYLA, France) used at a dilution of 1:250.The secondary antibodies were fluorescene conjugated anti-mouse oranti-rabbit. Antibodies were incubated with cells for 1 hour each.Between each incubation, the cells were washed three times for 5 minuteseach with PBS. Nuclear counterstain was done using DAPI. Dapi stainingto detect nucleus was done in 1:10,000 dilution in PBS. It was incubatedfor 5 minutes, followed by three washes for 5 minutes each in PBS.Photographic exposure times and contrast enhancements were identical forthe infected cells and control cell images.

Exposure of cells to an anti-core antibody demonstrated the presence ofHCV core protein in vMK0-Z infected cells. Exposure of cell to ananti-zeocin resistance protein demonstrated the presence of the Zeocinresistance protein in vMK0-Z infected cells.

Example 8 Construction of Subgenomic and Genome-Length Dicistronic RNAs

This example demonstrates the successful construction of replicationcompetent, selectable dicistronic replicons from an infectious clone ofa Japanese genotype 1b HCV virus (HCV-N) (Beard et al., Hepatol., 30,316-324, (1999)). Unlike other replicons, adaptive mutations are notrequired for efficient replication of these HCV-N replicons in Huh7cells or for the selection of Huh7 clones under G418 selection. We alsodemonstrate the replication competence of similar selectable,dicistronic RNAs incorporating the NS2-NS5B, E1-NS5B, or completecore-NS5B sequences of this virus. Our findings extend the range ofreplication competent HCV replicons to a second, genotype 1b virus andshow that a natural 4-amino-acid insertion within the NS5A protein ofthe wild-type HCV-N virus has a controlling role in determining thereplication capacity of this RNA in cultured Huh7 cells.

Materials and Methods

Plasmids.

The plasmid pBNeo/3-5B (FIG. 13) contains the Con1 sequence of theI₃₇₇neo/NS3-3′ replicon of Lohmann et al. (Lohmann et al., Science, 285,110-113 (1999), GenBank accession no. AJ242652) downstream of the T7promoter which is present in the vector upstream of the 5′ untranslatedregion (FIG. 13) (obtained from M. Murray, Schering-Plough ResearchInstitute, Kenilworth, N.J.). pNNeo/3-5B (FIG. 13) contains the sequenceof a similar HCV replicon in which almost all of the NS3-NS5B sequenceof the 3′ cistron is derived from an infectious molecular clone of thegenotype 1b virus, HCV-N (GenBank accession no. AF139594) (Beard et al.,Hepatol., 30, 316-324, (1999)). It was constructed by replacing thelarge BsrGI-XbaI fragment of pBNeo/3-5B with the analogous HCV sequencederived from the plasmid pHCV-N. This fragment swap results in theNS3-NS5B sequence in pNNeo/3-5B being identical to that of HCV-N, withthe exception of substitutions at 2 amino acid residues that retain theCon1 sequence: a Lys-to-Arg substitution at residue 1053 and anAla-to-Thr substitution at residue 1099 (where the numbering system isbased on the location within the original full length polyprotein asdescribed at GenBank AF139594), near the N-terminus (proteinase domain)of the NS3 protein. The 5′ untranslated region (′UTR) and N-terminalcore protein sequences of HCV-N and the BNeo/3-5B replicon areidentical.

The mutant pNNeo/3-5BΔi5A (FIG. 13) was derived from pNNeo/3-5B by anin-frame deletion removing a unique 4-amino-acid insertion that ispresent in the NS5A sequence of HCV-N in comparison to the consensusgenotype 1b sequence (Beard et al., Hepatol., 30, 316-324, (1999)). Thiswas accomplished by QuickChange mutagenesis (Stratagene, La Jolla,Calif.). By similar methods, additional mutations were created withinthe background of pNNeo/3-5B and pNNeo/3-5BΔi5A incorporatingsingle-amino-acid substitutions within NS5A or NS5B that have previouslybeen reported to enhance the replication capacity of the 1377/NS3-3′replicon (BNeo/3-5B) by others: the R2884G mutation described by Lohmannet al. (J. Virol., 75, 1437-1449 (2001)), and the S11791 mutationdescribed by Blight et al. (Blight et al., Science, 290, 1972-1974(2000)). These mutations are referred to as R2889G and S2005I,respectively, for the purposes of this study, according to the locationof these residues within the original full-length HCV-N polyproteinsequence. The resulting mutants were designated NNeo/3-5B(RG) andNNeo/3-5B(SI). Similar substitutions were introduced into the backgroundof pBNeo/3-5B to generate BNeo/3-5B(RG) and BNeo/3-5B(SI). Twoadditional mutants, NNeo/3-5BΔGDD and BNeo/3-5BΔGDD, each possess anin-frame deletion of 10 amino acids (MLVNGDDLVV) spanning the GDD motif(underlined) within the NS5B RNA-dependent RNA polymerase of bothwild-type replicons. DNA sequencing of the manipulated regions of theplasmids verified all mutations.

Selectable, dicistronic replicons containing part or all of the HCV-Nstructural protein-coding sequence within the 3′ cistron were generatedas follows. The plasmid pNNeo/C-5B contains the full-length HCV-Npolyprotein-coding sequence downstream of the EMCV IRES (see FIG. 14).To construct it, DNA fragments representing the EMCV IRES and HCV coreprotein-coding sequence were fused by overlapping PCR. Briefly, theprimer set to amplify the EMCVIRES-core fusion were as follows. For EMCVand part of core sequence containing fragment, sense primer,5′-TCCCTCTAGA CGGACCGCTA TCAGGACATA GC (SEQ ID NO:43) (which correspondsto nucleotides 1030-1051 of 1377/NS3-3′UTR (AJ242652), within the EMCVcoding region, and italics indicate non HCV replicon sequence) andantisense primer, 5′-ATTCGTGCTC ATGGTATTAT CGTGTTTTTC AAAGG (SEQ IDNO:44) (where the italicized nucleotides correspond to nucleotides342-353 of HCV-N, and the remainder correspond to nucleotides 1778-1800of 1377/NS3-3′UTR. For part of the EMCV and core containing fragment;the sense primer was 5′-CACGATAATA CCATGAGCAC GAATCCTAAA CCTC (SEQ IDNO:45), which corresponds to nucleotides 1789-1800 of 1377/NS3-3′UTR(AJ242652) within EMCV coding region, and italics indicate HCV N corecoding region nucleotides 342-363) and antisense primer, 5′-CCGCTCGAGGCAGTCGTTCG TGACATGGTA TACC (SEQ ID NO:46) (italics indicate non HCVreplicon nucleolides, and the remainder correspond to nucleotides938-962 of HCV-N). The resulting DNA was digested with RsrII and BstZ17Iand then ligated with the XbaI-RsrII fragment of pBNeo/3-5B and theBsIZ17I-XbaI fragment of pHCV-N.

pNNeo/E1-5B contains sequence encoding the C-terminal 22 amino acids ofthe core protein, the downstream E1 and E2 sequences and the remainderof the HCV-N polyprotein coding sequence. To construct it, a DNAfragment containing the EMCV sequence was fused to the E1 sequence by anoverlapping PCR. Briefly, the primer set to amplify the EMCVIRES-E1fusion were as follows. For EMCV and part of the E1 containing fragment,the sense primer was 5′-TCCCTCTAGA CGGACCGCTA TCAGGACATA GC (SEQ IDNO:47) (which corresponds to nucleotides 1030-1051 of 1377/NS3-3′UTR(AJ242652), within EMCV coding region, and italics indicate non HCVreplicon nucleotides) and antisense primer, 5′-AGAGCAACCG GGCATGGTATTATCGTGTTT TTCAAAGG (SEQ ID NO:48) (where italics correspond to E1sequence (nucleotides 849-861 of HCV-N) and the remaining nucleotidescorrespond to nucleotides 1778-1803 of 1377/NS3-3′UTR. For part of theEMCV and E1 containing fragment; the sense primer was 5′-CACGATAATACCATGCCCGG TTGCTCTTTT TCTATCTTCC (SEQ ID NO:49) (which corresponds tonucleotides 1789-1803 of 1377/NS3-3′UTR (AJ242652), within EMCV codingregion, and italics indicate nucleotides 849-873 of the HCV N E1) andantisense primer, 5′-ATGTACAGCC GAACCAGTTG CC (SEQ ID NO:50) (whichcorresponds to nucleotides 1983-2004 of HCV-N). The resulting DNA wasdigested with RsrII and NotI, and then ligated to the XbaI-RsrIIfragment of pBNeo/3-5B and NotI-XbaI fragment of pHCV-N.

The 3′ cistron of pNNeo/2-5B contains sequence encoding the NS2-NS5Bproteins of HCV-N, immediately downstream of the EMCV IRES. It wasconstructed in a fashion similar to pNNeo/C-5B and pNNeo/E1-5B, withfusion of the EMCV and NS2 sequences by an overlapping PCR. Briefly, theprimer set to amplify the EMCVIRES-NS2 fusion were as follows. For EMCVand part of the NS2 sequence containing fragment, the sense primer was5′-TCCCTCTAGA CGGACCGCTA TCAGGACATA GC (SEQ ID NO:51) (which correspondsto nucleotides 1030-1051 of 1377/NS3-3′UTR (AJ242652), within EMCVcoding region, and italics indicate non HCV replicon sequence) andantisense primer, 5′-CTCCCGGTCCATGGTATTAT CGTGTTTTTC AAAGG (SEQ IDNO:52) (where the italics indicate NS2 sequence of HCV-N (nucleotides2772-2783) and the remainder of the sequence corresponds to nucleotides1778-1800 of 1377/NS3-3′UTR. For part of the EMCV and NS2 containingfragment; the sense primer was 5′-CACGATAATA CCATGGACCG GGAGATGGCT GC(SEQ ID NO:53) (which corresponds to nucleotides 1789-1800 of1377/NS3-3′UTR (AJ242652), within EMCV coding region, and italicsindicate nucleotides 2772-2791 of the HCV-N NS2) and antisense primer,5′-GAGCGGTCCG AGTATGGCAA TCAG (SEQ ID NO:54) (which corresponds tonucleotides 3018-3041 of HCV-N). The resulting DNA was digested withRsrII and EcoRV, and ligated to the XbaI-RsrII fragment of pBNeo/3-5Band EcoRV-XbaI fragment from pHCV-N.

Cells

Huh7 cells were cultured in Dulbecco's modified Eagle's medium(Gibco-BRL, Invitrogen Life Technologies, Carlsbad, Calif.) supplementedwith 10% fetal calf serum, penicillin, and streptomycin. Transfectedcells supporting the replication of HCV replicons were maintained in thepresence of 1 mg of G418 (Geneticin) per ml and passaged two or threetimes per week at a 4:1 split ratio. In vitro transcription andtransfection of synthetic RNA.

Plasmid DNAs were linearized by XbaI and purified by passage through acolumn (PCR Purification Kit; Qiagen, Valencia, Calif.) prior totranscription. RNA was synthesized with T7 MEGAScript reagents (Ambion,Austin, Tex.) following the manufacturer's suggested protocol, and thereaction was stopped by digestion with RNase-free DNase. Followingprecipitation with lithium chloride, RNA was washed with 75% ethanol anddissolved in RNase-free water. For electroporation, Huh7 cells werewashed twice with ice-cold phosphate-buffered saline (PBS) andresuspended at 10⁷ cells/ml in PBS. RNA (1 to 10 μg) was mixed with 500μl of the cell suspension in a cuvette with a gap width of 0.2 cm(GenePulser II System; Bio-Rad, Hercules, Calif.). The mixture wasimmediately subjected to two pulses of current at 1.5 kV, 25 μF, andmaximum resistance. Following 10 minutes (min) of incubation at roomtemperature, the cells were transferred into 9 ml of growth medium andthe number of viable cells assessed by staining with trypan blue. Cellswere seeded into 10-cm-diameter cell culture dishes. For selection ofNeo-expressing cells, the medium was replaced with fresh mediumcontaining 1 mg of G418 per ml after 24 to 48 hours (h) in culture.

Indirect Immunofluorescence.

Cells were grown on chamber slides until 70 to 80% confluent, washedthree times with PBS, and fixed in methanol-acetone (1:1 [vol/vol]) for10 min at room temperature. Dilutions of primary, murine monoclonalantibodies to residues 1 to 61 of the core protein (MAB7013; MaineBiotechnology Services, Portland) (1:25), E2 (obtained from Y. Matsuuraand T. Miyamura, National Institute of Health, Tokyo, Japan) (1:400), orNS5A (MAB7022P; Maine Biotechnology Services) (1:10) were prepared inPBS containing 3% bovine serum albumin and incubated with fixed cellsfor 2 h at room temperature. After additional washes with PBS, specificantibody binding was detected with a goat anti-mouse immunoglobulinG-fluorescein isothiocyanate-conjugated secondary antibody(Sigma-Aldrich, St. Louis, Mo.) diluted 1:70. Cells were washed withPBS, counterstained with 4, 6-diamidino-2-phenylindole (DAPI), andmounted in Vectashield mounting medium (Vector Laboratories, Burlingame,Calif.) prior to examination by a Zeiss AxioPlan2 fluorescencemicroscope.

Northern Analysis.

To minimize potential variation in the intracellular abundance of HCVRNAs that might occur due to variation in the growth status of cells,RNA was extracted from freshly plated cultures after cells had reached70 to 80% confluence. Total cellular RNAs were extracted with TRIzolreagent (Gibco-BRL) and quantified by spectrophotometry at 260 nm. RNAswere separated by denaturing agarose-formaldehyde gel electrophoresisand transferred to positively charged Hybond-N+ nylon membranes(Amersham-Pharmacia Biotec, Piscataway, N.J.) with reagents providedwith the NorthemMax kit (Ambion) and the manufacturer's suggestedprotocol. RNAs were immobilized on the membranes by UV cross-linking(Stratagene) and stained with ethidium bromide to locate 28S rRNA on themembrane. The upper part of the membrane containing HCV replicon RNA(size greater than 28S) was hybridized with a digoxigenin-labeled,negative-sense RNA riboprobe complementary to the NS5B sequence ofHCV-N, while the lower part of the membrane containing P-actin mRNA washybridized with a digoxigenin-labeled, P-actin-specific riboprobe. Fordetection of the bound riboprobes, membranes were incubated withantidigoxigenin-alkaline phosphatase conjugate, reacted with CSPD (RocheMolecular Biochemicals, Indianapolis, Ind.), and exposed to X-ray film.

RT-PCR Amplification and Sequencing of cDNA from Replicating HCVRNAs.

Total cellular RNA was extracted from replicon-bearing cell lines asdescribed above and used as a template for the amplification of cDNAfragments spanning the NS3-NS5B segment of the NNeo/3-5B replicon.Reverse transcription (RT) was carried out with 1 μg of RNA, 200 U ofSuperScript II reverse transcriptase (Gibco-BRL), and two HCV-specificprimers (N6700R, 5′-AGCCTCTTCAGC AGCTG (SEQ ID NO:55) and N9411R5′-AGGAAATGGCCTATTGGC (SEQ ID NO:56), 1 μM), complementary to sequencein the NS4B and 3′UTR segments of the genome, in a total reaction volumeof 10 μl for 60 min at 42° C. cDNAs were subsequently amplified with PfuTurbo DNA polymerase (Stratagene) by 30 PCR cycles involving annealingat 60° C. for 60 seconds (s), extension at 72° C. for 120 s, anddenaturation at 95° C. for 30 s, followed by a final extension reactionat 72° C. for 2 min. Eight separate PCR primer sets were used to amplifynested segments spanning the NS3-NS5B region of the genome (see Table4). TABLE 4 Primer pairs. Primer sequence Corresponds to:TTTCCACCATATTGCCGTC  (SEQ ID NO: 57) nucleotides 1307-1325 of1377/NS3-3′UTR TTGACGCAGGTCGCCAGG   (SEQ ID NO: 58) nucleotides3551-3568 of HCV-N GAACCAGGTCGAGGGGGAGG (SEQ ID NO: 59) nucleotides3499-3519 of HCV-N TCGATGGGGATGGCTTTGCC (SEQ ID NO: 60) nucleotides4473-4492 of HCV-N CTCGCCACCGCTACGCCTCC (SEQ ID NO: 61) nucleotides3551-3568 of HCV-N ACTCCGCCTACCAGCACCC  (SEQ ID NO: 62) nucleotides5323-5341 of HCV-N ACCCCATAACCAAATACATC (SEQ ID NO: 63) nucleotides5260-5279 of HCV-N AGCCTCTTCAGCAGCTG    (SEQ ID NO: 64) nucleotides6207-6223 of HCV-N TATGTGCCTGAGAGCGACGC (SEQ ID NO: 65) nucleotides6144-6163 of HCV-N TATGTGCCTGAGAGCGACGC (SEQ ID NO: 66) nucleotides7116-7132 of HCV-N AACCTTCTGTGGCGGCAGG  (SEQ ID NO: 67) nucleotides7044-7062 of HCV-N CTGGTTGGACGCAGAAAACC (SEQ ID NO: 68) nucleotides8042-8061 of HCV-N AACCACATCCGCTCCGTGTG (SEQ ID NO: 70) nucleotides7962-7981 of HCV-N TGGCTCAATGGAGTAACAGG (SEQ ID NO: 71) nucleotides8962-8981 of HCV-N TTCTCCATCCTTCTAGCT   (SEQ ID NO: 72) nucleotides8901-8918 of HCV-N AACAGGAAATGGCCTATTG  (SEQ ID NO: 73) nucleotides9412-9431 of HCV-NThe sequence of each amplified cDNA segment was determined directly withan ABI 9600 automatic DNA sequencer. The existence of mutations wasconfirmed by sequencing the products of at least two separate RT-PCRs.ResultsAutonomous Replication of Subgenomic HCV Replicons Derived from HCV-N

HCV-N is a genotype 1b virus (Beard et al., Hepatol., 30, 316-324,(1999)) that shares only about 90% nucleotide identity in the NS3-NS5Bregion with the Con1 sequence present in the replicon RNAs described byLohmann et al. (Lohmann et al., Science, 285, 110-113 (1999)) and Blightet al. (Science, 290, 1972-1974 (2000)). To determine whether subgenomicRNAs derived from a previously constructed molecular clone of this virusare capable of replication in Huh7 cells, a plasmid was constructed witha T7 transcriptional unit containing the sequence of a candidatereplicon, NNeo/3-5B (FIG. 13). The organization of RNA transcriptsgenerated from this plasmid is identical to that of the I₃₇₇neo/NS3-3′replicon of Lohmann et al. (Lohmann et al., Science, 285, 110-113(1999)) (designated BNeo/3-5B in this study), with the 5′UTR of HCV andimmediately downstream sequence encoding the N-terminal 12 amino acidsof the core protein fused in-frame to the selectable marker, Neo,followed by the IRES of EMCV fused to the NS3-coding sequence anddownstream regions of the HCV genome, including the 3′UTR (FIG. 13). Thesequences of the proteins expressed by both the 5′ and 3′ cistrons ofNNeo/3-5B are identical to those of HCV-N, with the exception ofsubstitutions at 2 amino acid residues near the amino terminus of NS3, aLys-to-Arg substitution at residue 1053 and an Ala-to-Thr substitutionat residue 1099. These substitutions derive from the Con1 sequenceemployed in construction of this plasmid.

In initial experiments, NNeo/3-5B transcripts were transfected into Huh7cells, and the cells were grown in the presence of G418 to select cellswith active expression of Neo from replicon RNAs undergoingamplification. BNeo/3-5B transcripts were transfected in parallel.Numerous G418-resistant cell colonies survived the selection process inHuh7 cultures transfected with NNeo/3-5B RNA, with the number of cellcolonies isolated proportional to the quantity of RNA electroporatedinto the cells. However, there were no surviving G418-resistant cellcolonies following transfection of NNeo/3-5BAGDD, a mutated repliconcontaining an in-frame deletion spanning the GDD motif in the NS5BRNA-dependent RNA polymerase. The absence of surviving cell coloniesfollowing transfection of this RNA indicates that amplification of theNNeo/3-5B replicon is essential for 6418 resistance. Despitereproducible isolation of greater than 1,000 colonies from culturestransfected with 1 μg of NNeo/3-5B RNA, we were unable to isolate anycolonies from cells transfected with an equivalent quantity of eitherBNeo/3-5B or BNeo/AGDD RNA. The failure to recover G418-resistantcolonies following transfection of BNeo/3-5B suggests strongly that thispreviously described RNA replicates significantly less efficiently thanNNeo/3-5B in these Huh7 cells.

To confirm the presence of replicating subgenomic RNAs in cells selectedfor G418 resistance following transfection with NNeo/3-5B, threeG418-resistant cell colonies were selected at random and clonallyisolated. These clonal cell lines were then examined for the presence ofHCV RNA by Northern analysis. The presence of a substantial abundance ofHCV-specific RNA with a length approximating 8 kb was detected inextracts of total cellular RNA prepared from each of these stable celllines (data shown only for clones 1 and 2). Although the abundance ofthe replicon RNA was significantly greater in the BNeo/3-5B(RG) cellline than in other cell lines studied in this particular experiment, wenoted no consistent trends in the abundance of replicon RNA among celllines derived with different replicon constructs. Abundant NS5A proteinwas also demonstrated in each of the cell lines by indirectimmunofluorescence. These data confirm the ability of wild-type HCV-Nsubgenomic replicons to undergo autonomous replication in Huh7 cells andrepresent an important confirmation of the results of Lohmann et al.(Lohmann et al., Science, 285, 110-113 (1999)) with a second,independent isolate of HCV.

Adaptive Mutations are not Required for Efficient Replication ofNNeo/3-5B RNA.

Data reported both by Lohmann et al. (J. Virol., 75, 1437-1449 (2002))and by Blight et al. (Science, 290, 1972-1974 (2000)) suggest thatspontaneously arising, cell culture-adaptive mutations are required forefficient replication of BNeo/3-5B in Huh7 cells. Such mutations appearto be present within each replicon-bearing cell line that has beenclonally isolated and characterized in detail (Blight et al., Science,290, 1972-1974 (2000), Krieger et al., J. Virol., 75, 4614-4624 (2001),Lohman et al., J. Virol., 75, 1437-1449 (2002)). Cell culture-adaptivemutations have been identified within NS3, NS5A, and NS5B and have beenshown to dramatically increase the efficiency of colony formation whencells are transfected and subjected to G418 selection. To determinewhether such adaptive mutations are also required with NNeo/3-5Breplicons derived from HCV-N, we determined the nucleotide sequences ofthe NS3-NS5B segment of the replicons present in the three clonal celllines described in the preceding section. RNA extracted from these cellswere reverse transcribed into cDNA and amplified by RT-PCR for directDNA sequencing as described in Materials and Methods.

Replicon RNAs in two of the three cell lines contained single-amino-acidmutations: a 3-base insertion resulting in a new Lys residue at position2040 (NSSA) in clone 2, and a single-base change leading to a Cys-to-Sersubstitution at residue 1519 (NS3 helicase domain) in clone 3.Remarkably, there were no mutations identified in the amino acidsequence of the nonstructural proteins in clone 1, despite the fact thatthe replicon RNA abundance in these cells was approximately equivalentto that in other G418-resistant cell lines, including clone 2, in whichthere was the insertion of an additional residue in NS5A. These resultsconfirm that NNeo/3-5B RNA is capable of efficient autonomousreplication in the absence of adaptive mutations and suggest that thetwo mutations may have relatively little impact on the replication ofthis RNA.

Effect of BNeo/3-5B Adaptive Mutations on Replication of NNeo/3-5B.

To determine whether mutations in NS5A or NS5B that have been reportedpreviously to enhance the replication of BNeo/3-5B would further enhancethe replication of NNeo/3-5B replicons, we constructed NNeo/3-5B-derivedreplicons with a Ser-to-Ile substitution at residue 2005, NNeo/3-5B(SI),comparable to the Con1 replicons containing the S117931 mutation in NS5Adescribed by Blight et al. (Science, 290, 1972-1974 (2000)), or anArg-to-Gly substitution at residue 2889, NNeo/3-5B(RG), comparable tothe replicon containing the R2884G mutation in NS5B reported by Lohmannet al. (J. Virol., 75, 1437-1449 (2002)). Identical mutations were alsointroduced into BNeo/3-5B, leading to the creation of BNeo/3-5B(SI) andBNeo/3-5B(RG), respectively, and the modified NNeo/3-5B and BNeo/3-5BRNAs were transfected into Huh7 cells in parallel experiments.

The results of these experiments confirmed the cell culture adaptiveactivities of these NS5A and NS5B mutations on Con1-derived replicons.The introduction of S20051 into the background of BNeo/3-5B increasedthe efficiency of G418-resistant colony formation substantially morethan the introduction of R2884G. The number of colonies generatedfollowing transfection of Huh7 cells with BNeo/3-5B(SI) RNA approximatedthat obtained with NNeo/3-5B RNA. These results thus confirmed theimportance of the S20051 substitution for replication of the BNeo/3-5Breplicon, as reported previously (Blight et al., Science, 290, 1972-1974(2000)). However, they also demonstrated that the wild-type NNeo/3-5BRNA is comparable to BNeo/3-5B RNAs containing adaptive mutations suchas S20051 in terms of its ability to replicate in Huh7 cells and lead tothe selection of G418-resistant colonies. In fact, there was no apparentdifference in the abundance of HCV RNA in cell lines selected followingtransfection of BNeo/3-5B(SI) and NNeo/3-5B (clone 1, which contains noadaptive mutations). Interestingly, however, a cell line selectedfollowing transfection with BNeo/3-5B(RG) had a greater abundance ofviral RNA despite the substantially lower number of 6418-resistant cellcolonies generated with this RNA. We did not determine whether thisparticular cell line contained additional adaptive mutations.

The introduction of either of these two mutations into the background ofNNeo/3-5B also resulted in an increase in the number of G418-resistantcolonies, but proportionately this increase was much less than thatobserved with the introduction of these mutations into the BNeo/3-5Bbackground. The S20051 and R2889G mutations resulted in comparableincreases in the numbers of G418-resistant colonies, although thedensity of colony formation made their enumeration difficult even whenonly 1 μg of RNA was transfected per culture dish. However, we alsocompared the effects of these two mutations when introduced into thebackground of a similar subgenomic HCV-N replicon containing blastocidinrather than Neo as a selection marker (NBla/3-5B). In this case, whereblastocidin is generally less efficient than Neo as a selectable marker,the introduction of R2889G was shown to result in an ˜5-fold highernumber of G418-resistant cell colonies than the introduction of S20051.Importantly, the introduction of these mutations increased the number ofG418-resistant colonies obtained with NNeo/3-5B replicons no more thanseveralfold, and far less than the 1,000-fold or greater increases seenwith the comparable BNeo/3-5B replicons. Neither mutation resulted in anincrease in the abundance of replicon RNA in G4] 8-resistant cell linesselected following transfection with NNeo/3-5B RNAs.

Enhanced Replication Capacity of HCV-N RNA is Due to a Natural4-amino-acid Insertion in NS5A.

As mentioned above, the sequence of the infectious HCV-N cDNA clonecontains a unique 4-amino-acid insertion (-Ser-Ser-Tyr-Asn-) within theISDR segment of the NS5A protein in alignments with other HCV sequences(Beard et al., Hepatol., 30, 316-324, (1999)). This insertion includesamino acid residues 2220 to 2223 in the HCV-N polyprotein and, althoughunique in the database, was present in cDNA cloned directly from theJapanese patient who served as the source of the HCV-N isolate (Hayashiet al., J. Hepatol., 17, S94-S107 (1993)). It is thus representative ofthe wild-type sequence of this virus. Since mutations that enhance thereplication of the BNeo/3-5B replicon have been suggested to clusternear the ISDR of NS5, we questioned whether the presence of this uniqueinsertion in the ISDR might contribute to the ability of NNeo/3-5Breplicons to replicate efficiently in the absence of additional cellculture-adaptive mutations. To address this question, we deleted the4-amino-acid insertion from NNeo/3-5B (generating NNeo/3-5BΔi5A) andassessed the ability of this NS5A deletion mutant to support theselection of 6418-resistant colonies following transfection of Huh7cells. Additional deletion mutants were generated by removal of the4-amino-acid insertion from NNeo/3-5B(SI) and NNeo/3-5B(RG), designatedNNeo/3-5B(SI) iSA and NNeo/3-5B(RG) iSA, respectively.

The number of G418-resistant colonies selected following transfectionwith NNeo/3-5BΔi5A was much lower than after transfection withNNeo/3-5B. Only a small number of colonies were generated followingtransfection with a large amount of RNA (20 μg per culture dish),confirming the importance of this insertion to replication of this RNAin Huh7 cells. In contrast, the deletion of these 4 amino acids from theNS5A sequences of NNeo/3-5B(SI) resulted in only a modest decrease inthe efficiency of colony formation, with large numbers of G418-resistantcolonies selected after transfection of relatively small amounts ofNNeo/3-5B(SI) i5A RNA (1 μg/culture dish). Similar results were obtainedwith the NNeo/3-5B(RG) i5A replicon, although the number of survivingG418-resistant colonies was less than that with NNeo/3-5B(SI). The factthat efficient G418-resistant colony-forming activity could be preservedby either of these previously described cell culture adaptive mutationsin the absence of the 4-amino-acid insertion in NS5A provides furtherevidence that the 4-amino-acid insertion is responsible for the inherentability of NNeo/3-5B RNA to replicate efficiently in Huh7 cells.

Since many of the mutations that enhance the replication of BNeo/3-5Bhave been localized to the NS5A sequence (Blight et al., Science, 290,1972-1974 (2000), 14), we compared the NS5A sequences of NNeo/3-5B andBNeo/3-5B. The proteins are predicted to differ at 49 of 451 (11%) aminoacid residues (FIG. 15). Amino acid differences are scattered across thelength of the protein sequence, although they are somewhat more frequentwithin the ISDR and C-terminal half of the protein. Interestingly, thereare no differences at any of the residues at which single-amino-acidsubstitutions have previously been reported to enhance the replicationcapacity of BNeo/3-5B.

The most striking difference in the NS5A sequences of these replicons isthe presence of the 4-amino-acid insertion within the ISDR of NNeo/3-5B.This insertion and, in fact, the entire ISDR are within a 47-amino-acidsegment that was shown to have been spontaneously deleted in a cell linebearing a BNeo/3-5B replicon isolated by Blight et al. (Science, 290,1972-1974 (2000)). This large deletion mutation significantly increasedthe numbers of G418-resistant cell colonies selected followingtransfection of BNeo/3-5B RNA (Blight et al., Science, 290, 1972-1974(2000)). When the 4-amino-acid insertion was deleted from NNeo/3-5B, itscapacity to generate G418-resistant colonies was substantially, althoughnot completely, eliminated. However, the ability of the RNA toefficiently generate G418-resistant colonies was preserved byintroduction of the BNeo/3-5B-adaptive S20051 mutation in NS5A and, to aslightly lesser extent, the R2889G mutation in NS5B. The 4-amino-acidinsertion in NS5A thus accounts, at least in part, for the uniqueability of the wild-type HCV-N RNA to replicate in these cells. It thusrepresents a natural cell culture-adaptive mutation. Although present inthe synthetic HCV-N RNA that gave rise to infection in a chimpanzee, asdescribed above (Beard et al., Hepatol., 30, 316-324, (1999)), thepersistence of this sequence polymorphism was not studied in thisanimal. Thus, it is not possible to comment further on its contributionto replication in vivo.

Replication Competence of Selectable Dicistronic HCV-N RNAs Encoding theStructural Proteins of HCV

Lohmann et al. (Lohmann et al., Science, 285, 110-113 (1999))demonstrated that subgenomic Con1 replicons containing the NS2-NS5Bsegment of HCV also were capable of autonomous replication in Huh7cells, although the number of G418-resistant colonies selected wassomewhat less than that obtained after transfection of cells withreplicon RNA containing only the NS3-NS5B segment. To determine whetherthe replication capacity of the HCV-N RNA would be influenced by theinclusion of NS2-coding sequence or sequences encoding the envelope andcore proteins of HCV-N, we constructed a series of plasmids withtranscriptional units encoding the selectable, dicistronic RNAs shown inFIG. 14. In addition to the NS3-NS5B coding sequence present inNNeo/3-5B, the 3′ cistrons of these dicistronic RNAs contain upstreamwild-type HCV-N sequence encoding NS2 (NNeo/2-5B), the envelope proteinsas well as NS2 (NNeo/E1-5B), or the entire polyprotein (NNeo/C-5B). RNAtranscripts prepared from these plasmids were transfected into Huh7cells, as described above, and in each case gave rise to G418-resistantcolonies after several weeks of culture in G418-containing media. Thenumber of colonies produced from each RNA diminished with the increasinglength of the second cistron, with ˜160 colonies obtained withNNeo/2-5B, ˜60 colonies with NNeo/E 11-5B, and only 22 colonies fromNNeo/C-5B. However, stable G418-resistant cell lines were clonallyisolated from transfections with each of these RNAs, indicating that theRNA remained replication competent despite the inclusion of theadditional sequence.

Total cellular RNA extracted from these G418-resistant cell lines wasanalyzed by Northern analysis for HCV RNA. Each cell line containedHCV-specific RNA of the appropriate length, confirming the ongoingreplication of HCV RNA in cell lines selected after transfection witheach of the RNAs shown in FIG. 14. However, cells selected followingtransfection with NNeo/C-5B contained a demonstrably lower abundance ofreplicon RNA than cells selected following transfection with NNeo/2-5Bor NNeo/E1-5B. These latter cell lines were comparable in repliconabundance to cells selected following transfection with NNeo/3-5B.Furthermore, 6418-resistant cells selected with the NNeo/C-5B replicongrew slowly and failed to become completely confluent after severalweeks in culture. Colonies of cells selected from one of the NNeo/C-5Bcell lines were subcloned and, after passage for an additional month,demonstrated improved growth properties. Northern analysis of totalcellular RNA extracted from three of these NNeo/C-5B subclones containedviral RNA of the appropriate length, with an abundance approximatingthat of replicon RNA in cell lines selected following transfection withNNeo/3-5B.

G418-resistant cell lines selected following transfection withNNeo/E1-5B or NNeo/C-5B were examined for the presence of structuralprotein antigens by indirect immunofluorescence. In addition to NS5Aantigen, cells selected following transfection with NNeo/E1-5B containeddetectable E2 antigen, while cells selected following transfection withNNeo/C-5B RNA stained positively for core antigen. In both cases, only aproportion of the cells present in the clonally isolated cell linescontained a detectable abundance of these antigens at any single pointin time. This result was different from what was observed withG418-resistant cell lines selected following transfection withNNeo/3-5B, in which almost all cells contained detectable NS5A antigen.It is possible that this may reflect cell cycle dependence of thereplication of these RNAs (Pietschmann et al., J. Virol., 75, 1252-1264(2001)), because the cell lines were clonally derived and stable.Together, however, these data provide strong confirmatory evidence ofthe replication competence of genome-length, selectable, dicistronicHCV-N RNAs in Huh7 cells.

Example 9 Subgenomic Hepatitis C Virus Replicons Inducing Expression ofa Secreted Enzymatic Reporter Protein

This Example describes a useful refinement of these subgenomic repliconsthat simplifies detection of HCV RNA replication in bothtransiently-transfected cells and established cell clones selected underantibiotic pressure. By modifying the upstream cistron so that itexpresses the tat protein of human immunodeficiency virus (HIV) inaddition to the Neo resistance marker, replicon RNAs were developed thatare capable of signaling their presence and abundance in cells by thesecretion of placental alkaline phosphatase (SEAP), expressed undertranscriptional control of the HIV LTR. This system permits theautonomous replication of the viral RNA to be monitored in intact cellsby an enzymatic assay of SEAP activity in the media bathing the cells.Using these novel reporter replicons, we show the effect of interferon-αon the replication of RNAs derived from two different strains of HCV instably transformed cell cultures.

Materials and Methods

Cells. En5-3 cells are a clonal cell line derived from Huh7 cells bystable transformation with the plasmid pLTR-SEAP (see below). Thesecells were cultured in Dulbecco's modified Eagle's medium (Gibco BRL)supplemented with 10% fetal calf serum, 2 μg/ml blasticidin(Invitrogen), penicillin and streptomycin. Following transfection withreplicon RNAs, cells supporting replicon amplification were selected andmaintained in the above media containing in addition 400 μg/ml G418(geneticin). Cell lines were passaged once or twice per week.

Plasmids. The plasmid pLTR-SEAP was generated as follows. pcDNA6/V5-His(Invitrogen) was digested with BglII-BamHI to remove the CMV promoter.The vector was then self-ligated, digested with EcoRV-NotI, andreligated to a DNA fragment encoding SEAP under transcriptional controlof the HIV LTR that was amplified from pBCHIVSEAP (obtained from B.Cullen, Duke University, Durham, N.C.) using the oligonucleotide primerpairs; 5′-CTAGCTAGCCTCGAGACCTGGAAAAACATGGAG (SEQ ID NO:8) and5′-ATAAGAATGCGGCCGCTTAACCCGGGTGCGCGG (SEQ ID NO:9). The resultingplasmid was transfected into Huh7 cells using a non-liposomaltransfection reagent (FUGENE, Boerhinger Manheim), and stably resistantcells were selected in the presence of blasticidin (Invitrogen).Blasticidin-resistant cell colonies were clonally selected and subjectedto further characterization. One, designated En5-3, was selected forsubsequent use due to a low basal level of SEAP activity and efficientinduction of SEAP following expression of the HIV tat protein.

To construct the plasmid pEt2AN, a DNA fragrnent containing the EMCVIRES was amplified by PCR from pEMCV-CAT (Whetter et al., Arch Viol.,136, 291-298 (1994)) using paired primers containing HindIII and Stulsites, respectively. DNA encoding the tat protein was similarlyamplified from pCTAT (also a generous gift of Dr. Cullen) with pairedprimers containing StuI and EcoRI sites, respectively. Finally, a DNAfragment encoding 15 amino acids of the foot-and-mouth disease virus(FMDV) 2A protein was generated by annealing the complementary primers5′-AATTCGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCTGGGC CCG (SEQ ID NO:24)and 5′-GATCCGGGCCCAGGGTTGGACTCGACGTCTCCCGCAAGCTTAAGAAG GCG (SEQ IDNO:74) to form a duplex DNA molecule with EcoRI and BamHI sticky ends,respectively. The neo sequence was amplified from pRcCMV (Invitrogen)with primer pairs containing BglII and NotI. These fragments wereligated to pcDNA6/V5-His (Invitrogen) digested with HindIII and NotI togenerate pEt2AN.

To construct the replicon plasmid pBACtat2Aneo, the genotype 1ainfectious clone, pCV-H77c (generously provided by Dr. Robert Purcell,National Institutes of Health, Bethesda, Md.) was digested with SphI andthe small fragment was religated. A single T to A nucleotide change wasengineered in this plasmid at nucleotide 444 of the HCV sequence of H77c(GenBank accession number AF011751) using QuickChange (Statagene)mutagenesis, generating a novel HpaI site at this position. Thisresulting plasmid was digested with HpaI and XbaI to generate a DNAfragment representing the HCV 1a 5′NTR and immediately downstreamsequence encoding the first 14 amino acids of the HCV polyprotein. Asecond DNA fragment representing the tat, 2A, and partial neo sequencewas excised from pEt2AN by digestion with StuI and SphI. Finally, theplasmid pBNeo/wt (FIG. 16), containing the sequence of the1377neo/NS3-3′ replicon of Lohmann et al. (obtained form Michael Murray,Schering-Plough Research Institute) was digested with SphI and XbaI togenerate a fragment representing the C-terminal neo sequence, EMCV IRES,and downstream elements of the HCV replicon. These three fragments wereligated to generate pBACtat2Aneo (FIG. 16), which contains the 5′NTR anddownstream 42 nts of core-coding sequence of the H77 strain of HCV(genotype 1a) and the NS3-5B and 3′NTR sequence of the Con1 strain ofHCV (genotype 1b). The plasmid pBtat2Aneo was generated by QuickChangemutagenesis of pBΔCtat2Aneo, with deletion of the 42 nucleotides ofcore-coding sequence and fusion of the tat sequence directly downstreamof 5′NTR of HCV. pNtat2Aneo was constructed by exchanging the largeBsrGI-XbaI fragment of pBtat2Aneo with the analogous HCV sequencederived from the plasmid pHCV-N resulting in replacment of most of theNS3-NS5B and 3′NTR sequence. A similar strategy was employed for theconstruction of variants of these replicon plasmids containing variouscell culture-adaptive mutations or a deletion of the GDD motif in theNS5B protein, as described in Example 8.

RNA transcription and transfection. RNA was synthesized with T7MEGAScript reagents (Ambion), after linearizing plasmids with XbaI.Following treatment with RNase-free Dnase to remove template DNA andprecipitation of the RNA with lithium chloride, the RNA was transfectedinto En5-3 cells. Transfection was done by electroporation, as describedpreviously. Briefly, 10 μg RNA was mixed with 5×10⁶ cells suspended in500 μl phosphate buffered saline, in a cuvette with a gap width of 0.2cm (Bio-Rad). Electroporation was with two pulses of current deliveredby the Gene Pulser 11 electoporation device (Bio-Rad), set at 1.5 kV, 25μF, and maximum resistance.

In vitro translation. In vitro transcribed RNA, prepared as describedabove, was used to program in vitro translation reactions in rabbitreticulocyte lysate (Promega). About 1 mg of each RNA, 2 μl of[³⁵S]-methionine (1,000 Ci/mmol at 10 mCi/ml), and 1 ml of an amino acidmixture lacking methionine were included in each 50 ml reaction mixture.Translation was carried out at 30° C. for 90 min. Translation productswere separated by SDS-PAGE followed by autoradiography or Phosphorlmager(Molecular Dynamics) analysis.

Northern analysis for HCV RNA. We seeded replicon-bearing cells into 6well plates at a density of 2×10⁵ cells/well, and harvested the RNA fromindividual wells at daily intervals. Total cellular RNAs were extractedwith TRizol reagent (Gibco-BRL) and quantified by spectrophotometry at260 nm. One half of the total RNA extracted from each well was loadedonto a denaturing agarose-formaldehyde gel, subjected to electrophoresisand transferred to positively-charged Hybond-N+ nylon membranes(Amersham-Pharmacia Biotec) using reagents provided with the NorthernMaxKit (Ambion). RNAs were immobilized on the membranes by UV-crosslinking.The membrane was hybridized with a [³²P]-labeled antisense riboprobecomplementary to the 3′-end of NS5B sequence (HCV nucleotides 8990-9275corresponding to GenBank accession number AF139594), and the hybridizedprobe was detected by exposure to X-ray film.

Indirect immunofluorescence analysis. Cells were grown on chamber slidesuntil 70-80% confluent, washed 3 times with PBS, and fixed inmethanol/acetone (1:1 V/V) for 10 min at room temperature. A 1:10dilution of a primary, murine monoclonal antibody to NS5A (MAB7022P,Maine Biotechnology Services) was prepared in PBS containing 3% bovineserum albumin, and incubated with the fixed cells for 1 hr at roomtemperature. Following additional washes with PBS, specific antibodybinding was detected with a goat anti-mouse IgG FITC-conjugatedsecondary antibody (Sigma) diluted 1:70. Cells were washed with PBS,counterstained with DAPI, and mounted in Vectashield mounting medium(Vector Laboratories) prior to examination by a Zeiss AxioPlan2fluorescence microscope.

Alkaline phosphatase assay. SEAP activity was measured in 20 μl aliquotsof the supernatant culture fluids using the Phospha-LightChemiluminescent Reporter Assay (Tropix), and the manufacturer'ssuggested protocol reduced 1/3 in scale. The luminescent signal was readusing a TD-20/20 Luminometer (Turner Designs, Inc.). In most time courseexperiments, the culture medium was replaced every 24 hrs. Thus, theSEAP activity measured in these fluids reflected the daily production ofSEAP by the cells.

Real-time quantitative RT-PCR anaysis of HCVRNA. Quantitative RT-PCRassays were carried out using TaqMan chemistry on a PRISM 7700instrument (ABI). For detection and quantitation of HCV RNA, we usedprimers complementary to the 5′NTR region of HCV (Takeuchi et al.,Gastroenterology, 116, 636-642 (1999)), with in vitro transcribed HCVRNA included in the assays as a standard. Results were normalized to theestimated total RNA content of the sample, as determined by theabundance of cellular GAPDH mRNA detected in a similar real-time RT-PCRassay using reagents provided with Taqman GAPDH Control Reagents (Human)(Applied Biosystems).

Sequence analysis of cDNA from replicating HCV RNAs. HCV RNA wasextracted from cells, converted to cDNA and amplified by PCR asdescribed previously (see Example 8). First-strand cDNA synthesis wascarried out with Superscript II reverse transcriptase (Gibco-BRL), andpfu-Turbo DNA polymerase (Stratagene) was used for PCR amplification ofthe DNA. The amplified DNAs were subjected to direct sequencing using anABI 9600 automatic DNA sequencer.

Interferon treatment of cell cultures. Selected replicon-bearing celllines were seeded into 12 well plates. The media was replaced 24 hrslater with fresh, G418 free media containing various concentrations ofrecombinant interferon-α2B ranging from 0 to 100 units/ml. The mediumwas subsequently completely removed every 24 hrs, the cells washed, andrefed with fresh interferon-containing media. SEAP activity was measuredin the media removed from the cells as described above.

Results

Tat-SEAP enzyme reporter system. The HIV tat protein is a potenttranscriptional transactivator of its LTR promoter element. Unlike mostknown eukaryotic transcriptional transactivators, tat functions via aninteraction with an RNA structure, the transactivation responsiveelement (TAR), rather than through interaction with DNA (Naryshkin etal., Biochemistry, 63, 189-503 (1998); Cullen, Cell, 93, 685-692(1998)). In the absence of tat, almost all RNA transcripts initiated bythe LTR promoter are terminated prematurely within ˜60-70 nucleotides ofthe start site. Tat acts to promote the efficient elongation ofpremature transcripts, thereby transactivating the transcription offunctional mRNAs from sequences placed under control of the HIV LTRpromoter. We have taken advantage of the small size of the tat protein,and the manner in which it functionally regulates the LTR promoter, todevelop a system in which a replication-competent, subgenomic HCV RNAexpressing tat induces the expression of secreted alkaline phosphatase(SEAP) placed under transcriptional control of the LTR in stablytransformed liver cells.

pEt2AN is an expression plasmid in which the HIV tat coding sequence isfused to sequence encoding the FMDV 2A proteinase and the positive,selectable marker neomycin phosphotransferase (Neo) (FIG. 16A). Thesmall FMDV 2A polypeptide sequence possesses autocatalytic activity(Ryan et al., EMBO J., 13, 928-933 (1994)), resulting in the scission ofthe peptide backbone at its C-terminus and the release of Neo. Thetranslation of this minipolyprotein is driven by the EMCV IRES sequencelocated just upstream of the protein coding sequence (FIG. 16A), whiletranscription is directed by a composite CMV/T7 promoter. We used thisplasmid to determine the level of SEAP expressed by stably transformedHuh7 cells (selected for blasticidin resistance) in which the SEAPsequence had been integrated under transcriptional control of the HIVLTR. SEAP activity was measured in the supernatant culture medium beforeand after transfection of the cells with pEt2AN. Results obtained withone clonally-isolated cell line, En5-3, are shown in FIG. 16B.

This cell line produced a minimal basal level of SEAP activity, whiletransfection of the cells with pEt2AN DNA led to an approximately 100fold increase in the secretion of SEAP into the medium in response totat expression (FIG. 16B). The secretion of SEAP from En5-3 cells beganto increase between 24 and 48 hrs after DNA transfection, and reachedmaximal levels at 72 to 96 hrs. In contrast, the transfection of En5-3cells with RNA transcribed in vitro from pEt2AN led to an immediateincrease in SEAP activity that was maximal when first assayed at 24 hrspost-transfection and subsequently decreased over time, reachingbackground levels 72 hours later (FIG. 16C). Since the cell culturemedium bathing these transfected cells was replaced at 24 hr intervalsin these experiments (see Materials and Methods), the SEAP activitymeasured at each time point reflected the amount of the reporter proteinsecreted into the medium over the preceding 24 hr period. The delay inSEAP secretion following DNA versus RNA transfection is likely torepresent the time required for RNA transcription to occur, while therapid decline of SEAP following RNA transfection reflects degradation ofthe transfected RNA and the tat protein translated from it. Theseencouraging results suggested that the expression of tat from areplicating subgenomic HCV RNA could provide a simple and usefulapproach to monitoring the presence and abundance of replicon RNA inEn5-3 cells.

Subgenomic HCVreplicons expressing tat. To test this hypothesis, weconstructed a plasmid with a transcriptional unit containing adicistronic, subgenomic HCV replicon similar to that reported originallyby Lohmann et al. (Science, 285, 110-113 (1999)), but in which the 5′cistron encodes the tat-2A-Neo minipolyprotein present in pEt2AN (FIG.16), fused in frame downstream of the N-terminal 14 amino acid residuesof the HCV core protein sequence (FIG. 17, BΔCtat2ANeo). The secondcistron in this replicon contained the NS3-5B segment of the Con1 HCVsequence placed under the translational control of the ECMV IRES, as inthe original HCV replicons (Lohmann et al., Science, 285, 110-113(1999)). We also constructed a variant in which the 5′ cistron containedno HCV protein-coding sequence, and in which HCV IRES-directedtranslation initiated at the tat coding sequence (FIG. 17, Btat2ANeo).To enhance the potential replication of these replicons in Huh7 cells,additional variants were engineered to contain the S2205I (SI) cellculture-adaptive mutation described by Blight et al. (Science, 290,1972-1974 (2000)), and the R2889G (RG) mutation described by Krieger etal. (J. Virol, 75, 4614-4624 (2001)), respectively (these mutations arenumbered according to the location of the cognate residue within theHCV-N sequence) (see Example 8) (FIG. 17).

Since the fusion of heterologous sequence directly downstream of the HCVIRES may reduce the ability of the HCV IRES to direct the internalinitiation of translation on a hybrid RNA (Reynolds et al., EMBO J., 14,6010-6020 (1995); Rijinbrand et al., RNA, 7, 585-597 (2001)), weevaluated the translational activity of these replicons by programmingrabbit reticulocyte lysates for translation with RNAs transcribed fromthese plasmids. The results of these experiments confirmed the activityof the FMDV 2A proteinase within the minipolyprotein, as protein speciesmigrating with the mobilities expected for both the unprocessedDCtat2ANeo and tat2ANeo precursor proteins, and the fully processed Neoprotein, were evident in SDS-PAGE gels of the translation products fromBΔCtat2ANeo and Btat2ANeo, respectively (FIG. 18A, lanes 2 and 3). Thetat2A cleavage product was not observed due to its small size. Theresults also suggested that the absence of the core protein-codingsequence in Btat2ANeo did in fact result in a significant reduction intranslation of the upstream cistron, as reflected in reduced quantitiesof Neo and the tat2ANeo precursor protein in lysate programmed withBtat2ANeo RNA (FIG. 18A, compare lane 3 with lane 2). In contrast, thequantity of NS3 produced from the downstream cistron was relativelyincreased in lysates programmed with Btat2ANeo RNA compared toBΔCtat2ANeo, suggesting that the reduction in the activity of the HCVIRES in the former RNA may have a complementary, beneficial effect onthe downstream EMCV IRES. This suggests that there may be intercistroniccompetition for translation factors between the HCV and EMCV IRESelements in these replicon RNAs, as noted previously with otherdicistronic RNAs (Whetter et al., J. Virol., 68, 5253-5263 (1994)).

We next assessed the activities of tat proteins expressed from theupstream cistron in the BΔCtat2ANeo and Btat2ANeo replicons (FIG. 17) intransient transfections of these replicon RNAs in En5-3 cells. SEAPactivity was monitored in the supernatant media at 72 hrspost-transfection, in the absence of Neo selection. The results of theseexperiments indicated that the tat protein was significantly less activewhen expressed as a fusion protein with the N-terminal 14 amino acidsegment of core (FIG. 318B, compare BΔCtat2ANeo, BΔCtat2ANeo(SI) andBΔCtat2ANeo(RG), with Btat2ANeo, Btat2ANeo(SI) and Btat2ANeo(RG) RNAS).Although the tat proteins expressed from these RNAs also have aC-terminal fusion with the FMDV 2A proteinase, this C-terminal fusiondoes not abrogate the transactivating activity of tat, as evidenced inthe experiments shown in FIGS. 16B and 16C. Replication of the RNAs didnot contribute to the expression of SEAP in the transient transfectionexperiment shown in FIG. 18B, as the amount of SEAP induced bytransfection of an NS5B deletion mutant, Btat2ANeo(ΔGDD), was onlyslightly less than that induced by its parent, Btat2ANeo. Similarly, thecell culture-adaptive NS5A S22051 and NS5B R2889G mutations (FIG. 17)engineered into these RNAs had no effect on the level of SEAP expressionunder these conditions (FIG. 18B).

Stable cell lines expressing SEAP under control of replicon-mediated tatexpression. Efforts to select stable, G418-resistant colonies followingtransfection of En5-3 cells with Btat2ANeo or BΔCtat2ANeo wereunsuccessful. These results are consistent with the very low frequencyof colony formation with the unmodified Con1 NS3-5B sequence, asreported by Lohmann and others (Lohmann et al., Science, 285, 110-113(1999); Blight et al., Science, 290, 1972-1974 (2000)).

However, it was possible to select G418-resistant En5-3 clones followingtransfection of the modified Btat2ANeo containing the adaptive S22051mutation and BΔCtat2ANeo RNAs containing the adaptive S2205I and R2889Gmutations in NS5A and NS5B (FIG. 17), respectively. The efficiency ofcolony formation was substantially lower with these replicons, even withthe adaptive mutations, than what has been reported in the literature(Lohmann et al., J. Virol., 75, 1437-1449 (2001); Blight et al.,Science, 290, 1972-1974 (2000)) or what we have observed previously (seeExample 8) with dicistronic, subgenomic HCV replicons. This may reflectthe use of the clonal, blastocidin-resistant En5-3 cell line rather thanthe parental Huh7 cells. Moreover, the number of colonies selected withBtat2ANeo(SI) RNA was approximately 10-fold lower than withBΔCtat2ANeo(SI), suggesting that the absence of the short, AC coreprotein-coding sequence in Btat2ANeo(SI) decreases the efficiency ofcolony selection. This could be due to the lower level of Neo expressedfrom this RNA (FIG. 18), or potentially to other effects on replicationof the subgenomic RNA.

Because replicons containing the genotype 1b, HCV-N sequence have provento be substantially superior to Con1 replicons in their ability toinduce the selection of G418-resistant Huh7 cell clones (see Example 8),we constructed a parallel series of replicons containing the tat2ANeosequence in the upstream cistron with the downstream cistron, NS3-NS5Bsequence derived from HCV-N: Ntat2ANeo, Ntat2ANeo(SI) and Ntat2ANeo(RG)(FIG. 17). Transfection with each of these RNAs led to the selection ofstable, G418-resistant colonies. The number of G418-resistant coloniesselected with Ntat2ANeo(RG) was at least 100-fold higher than withBtat2ANeo(SI). Overall, the efficiency of colony selection observed withreplicon RNAs that lacked any core protein coding sequence (FIG. 17)could be ordered as follows, from high to low: Ntat2ANeo(SI),Ntat2ANeo(RG), Ntat2ANeo, Btat2ANeo(SI). This is consistent with ourprevious observations with subgenomic HCV replicons expressing only Neofrom the upstream cistron (see Example 8).

Replicon RNA was readily detected by northern analysis of G418-resistantcell lines selected following transfection with BΔCtat2ANeo(SI),Btat2ANeo(SI) and Ntat2ANeo(RG) (FIG. 19A). The abundance of the viralRNA was significantly greater in the BΔCtat2ANeo(SI) cell line selectedfor testing, than in cell lines supporting replication of Btat2ANeo(SI)and Ntat2ANeo(RG). While the total abundance of the replicon RNAs (seeMaterials and Methods) increased in each of the cell lines studied overa 120 hr period following passage of the cells (FIG. 19A), quantitativereal-time RT-PCR assays showed a trend toward a reduction in theintracellular abundance of the replicon RNA relative to the abundance ofGAPDH mRNA as the cells approached confluence at 120 hrs (FIG. 19B).This is similar to the reduction in intracellular abundance of repliconRNAs reported recently by Pietschmann et al. (J. Virol., 75, 1252-1264(2001)). Once confluent, the intracellular abundance of the repliconRNAs appeared to be similar in all three cell lines studied. Theseresults confirm that there is no requirement for core-protein codingsequence for replication of these dicistronic, subgenomic viral RNAs.

We also examined the cell lines shown in FIG. 19 for viral proteinexpression as well as secretion of SEAP. NS5A antigen was readilydetected within the cytoplasm in each cell line, while no NS5A antigenwas detectable in normal En5-3 cells stained in parallel. The abundanceof the viral protein was significantly greater in cells containingBΔCtat2ANeo(SI) than Btat2ANeo(SI) or Ntat2ANeo(RG), consistent with thegreater abundance of replicon RNA detected in the former by northernanalysis (FIG. 19A). In contrast, the SEAP activities expressed by thesecell lines showed a very different relationship to the abundance of thereplicon RNA. Each of the cell lines secreted increased amounts of SEAPthat were detectable above the low background activity present in En5-3media (FIG. 20A). However, the level of SEAP activity expressed by theBΔCtat2A(SI) cell line was minimally above background and much lowerthan that secreted by the Btat2ANeo(SI) or Ntat2ANeo(RG) cell lines,despite a higher abundance of viral RNA and viral proteins in theformer. Sequencing of cDNA amplified by RT-PCR from the replicon RNAspresent in the BΔCtat2A(SI) cells did not identify any mutations withinthe upstream, ΔCtat2ANeo cistron, ruling out adventitious mutations as apotential cause for the minimal level of SEAP expressed by these cells.The Btat2ANeo(SI) and Ntat2ANeo(RG) cell lines demonstrated robustsecretion of the reporter protein, reaching levels at least 100-foldabove background after 5 days in culture (FIG. 20A). These results areconsistent with the results of the transient transfections presentedabove (FIG. 18B), and serve to confirm that the fusion of tat to theN-terminal segment of the core protein sharply diminishes its ability tofunctionally transactivate the HIV LTR.

In the experiment shown in FIG. 20A, it is important to note that themedia was completely replaced at 24 hr intervals, and that the cellswere thoroughly washed before being refed with fresh media. Thus, theresults shown represent the quantity of SEAP secreted by theBtat2ANeo(SI) and Ntat2ANeo(RG) cells during successive 24 hr periods.The secretion of SEAP correlated closely with the abundance of repliconRNA in the Btat2ANeo(SI) and Ntat2ANeo(RG) cells as determined bydensitometry of northern blots (FIG. 20B, R2=0.983 and 0.939 by linearregression analysis, respectively). In aggregate, these resultsdemonstrate that the expression of tat from subgenomic HCV RNAs that arereplicating in En5-3 cells effectively signals the secretion of SEAP,thereby providing an easily measurable and accurate marker of viral RNAreplication that does not require lysis or destruction of the cellmonolayer.

Impact of cell culture-adaptive mutations on the replication oftat-expressing HCV replicons in transient transfection assays. Furtherstudies of these replicons focused on those with no core proteinsequence fused to tat, since the fusion with the core sequenceeffectively inactivated the transactivating function of tat. Todetermine whether the activation of SEAP expression in En5-3 cells bytat was sufficiently sensitive for detection of the replication ofsubgenomic RNAs in transient transfection assays, replicon RNAs weretransfected into En5-3 cells using electroporation, and the cells werefollowed for a period of 20 days in the absence of G418 selection.Included in this experiment were the Btat2ANeo and Ntat2ANeo replicons,and mutants containing cell culture-adaptive mutations that were derivedfrom them, as shown schematically in FIG. 17B. The supernatant mediabathing the transfected cells was removed and replaced with fresh mediaat 24 hr intervals, as in the experiment shown in FIG. 20A, and thecells were collected by trypsinization and passaged into fresh culturevessels at 7 and 14 days. The levels of SEAP activity present in themedia that was removed from cells transfected with the replicon RNAsbased on the Btat2ANeo (Con1) sequence (FIG. 17) are shown in FIG. 21A,while FIG. 21B shows SEAP activities in media collected from cellstransfected with replicons derived from the HCV-N sequence.

The transfection of any of these replicon RNAs into En5-3 cells resultedin a high initial level of SEAP expression that was present in theculture media as early as 12 hrs after electroporation (FIGS. 21A and21B). This early, high level of SEAP secretion persisted forapproximately 3 days, and was due to translation of the transfectedinput RNA, as in the experiment shown in FIG. 18C. This high initialSEAP level was also observed with replication-defective mutantscontaining a deletion in the NS5B sequence involving the GDD polymerasemotif (ΔGDD mutants) (FIGS. 21A and 21B). The SEAP activity secretedinto the media of cells transfected with Btat2ANeo(ΔGDD) andNtat2ANeo(ΔGDD) began to decrease by day 4, and reached baseline valuessimilar to those observed with normal En5-3 cells by 8 days afterelectroporation (FIGS. 21A and 21B). In contrast, other, replicationcompetent RNAs, particularly those derived from the HCV-N sequence,demonstrated increased levels of SEAP expression at later time pointsthat were significantly above the En5-3 cell background and thusindicative of replication of the transfected RNA.

In experiments with replicon RNAs derived from the Con1 sequence,significant increases in SEAP activity above that observed with theBtat2ANeo(ΔGDD) mutant were seen only in cells transfected withBtat2ANeo(SI). There was no apparent difference in the levels of SEAPexpressed by cells transfected with the Btat2ANeo and Btat2ANeo(RG)replicons. Cells transfected with Btat2ANeo(SI) demonstrated a low levelbut sustained increase in SEAP activity above background beginning about10 days after transfection (FIG. 21A). However, the secretion of SEAPwas modest in magnitude, and never more than several-fold abovebackground. In sharp contrast, the HCV-N based replicons were remarkablymore potent in terms of their abilities to elicit sustained increases inSEAP expression (FIG. 21B). Levels of SEAP secretion up to 100-foldabove background were observed with Ntat2ANeo(SI) and Ntat2ANeo(RG), aswell as Ntat2ANeo(SIΔi5A). This latter replicon contains both the S2205Isubstitution in NS5A as well as the deletion of a natural 4 amino acidinsertion that is present in the NS5A sequence of HCV-N (FIG. 17B). Thisnatural insertion in NS5A, which was present in cDNA cloned from humanserum (Beard et al., Hepatology, 30, 316-324 (1999)), has been shown tocontribute substantially to the replication capacity of repliconscontaining the wild-type HCV-N sequence in Huh7 cells (Example 8). Theresults shown in FIG. 21 are consistent with those disclosed in Example8 concerning the relative abilities of subgenomic RNAs containing theCon1 and HCV-N NS3-NS5B sequences (with or without cell culture adaptivemutations in NS5A and NS5B) to transduce the selection of G418-resistantcell clones. These results also provide independent confirmation of theability of the S22051 and R2889G mutations to enhance the replicationcapacity of subgenomic, genotype 1b RNAs in cultured cells (Blight etal., Science, 290, 1972-1974 (2000); Krieger et al., J. Virol., 75,4614-4624 (2001); Example 8).

We also examined transiently transfected cells for expression of NS5Aantigen at 12 and 19 days after electroporation. These studiesdemonstrated that the proportion of cells containing a detectableabundance of NS5A was significantly greater following transfection withNtat2ANeo(RG) and Ntat2ANeo(SI), than Ntat2ANeo or Btat2ANeo(SI). Thus,these results parallel closely the results of the SEAP assays shown inFIG. 21. Interestingly, the intensity of staining of individual positivecells appeared similar with each of the replicon RNAs, suggesting thatthe level of SEAP expression may correlate with the proportion of cellsin which replicon amplification is occurring, rather than theintracellular abundance of the replicon under these conditions. As thisexperiment was carried out in the absence of G418 selection, it isuncertain whether those cells that did not stain positively for NS5Aantigen contained levels of the viral protein that were below thethreshold of detection or, alternatively, none at all.

Interferon suppression of HCV RNA replication. To demonstrate theutility of the tat-expressing HCV replicons, we assessed the ability ofrecombinant interferon-α2b to suppress the replication of Btat2ANeo(SI)and Ntat2ANeo(RG) in stable, G418 resistant cell clones. Recently seededcell cultures were fed with media containing various concentrations ofrecombinant interferon-α2B ranging from 0 to 100 units/ml. The mediumwas subsequently removed completely at 24 hr intervals, and the cellswere washed thoroughly and refed with fresh interferon-containing media.Results are shown in FIG. 22 and demonstrate dose-dependent inhibitionof SEAP secretion in both cell lines. As shown, cells cultured in theabsence of interferon, or at the lowest concentration of interferon,showed an increasing level of SEAP secretion over successive 24 hrintervals, consistent with the growth of the cells. At the highestconcentration of interferon tested (100 units/ml), this trend wasreversed and SEAP expression declined over time in the absence ofdemonstrable cellular cytotoxicity. Independent quantitative RT-PCRassays for HCV RNA demonstrated that the decline in SEAP secretion wasclosely matched by similar decreases in the intracellular abundance ofRNA (compare FIG. 22 and FIG. 23). The decline in intracellular RNApreceded the decreases in SEAP secretion by approximately 24 hrs, mostlikely reflecting the kinetic delay in tat signaling of SEAP secretion.

Surprisingly, the Ntat2ANeo(RG) replicon (FIG. 22B) was approximately10-fold more resistant to interferon than the Btat2ANeo(SI) replicon(FIG. 22A). This relative interferon resistance was reflected also indifferences in the degree of suppression of the intracellular abundanceof HCV RNA following interferon treatment of these cells (compare thedecrease in Btat2ANeo(SI) RNA abundance at different interferonconcentrations in FIG. 23A, with the decreases in Ntat2ANeo(RG) RNAabundance shown in FIG. 23B). A similar level of interferon resistancewas observed in separate experiments with an independently selected,G418-resistant clone supporting the replication of the Ntat2ANeo(RG)replicon, suggesting that the resistance observed in FIGS. 22B and 23Bwas not an idiosyncratic feature of the particular cell clone tested.Studies are currently in progress to determine the molecular basis ofthis difference in the response of the two replicons to interferon-α2b.

Discussion

We have described here an enzymatic reporter system that permits thedetection and quantitation of HCV RNA replication in intact cellmonolayers. The system is based on the expression of the tattransactivator protein by replicating subgenomic RNA replicons, and thesubsequent induction of SEAP synthesis in En5-3 cells that contain theSEAP gene under transcriptional control of the HIV LTR promoter. SEAP issecreted efficiently into the medium bathing these cells, where it isreadily quantified as an accurate marker of viral RNA abundance. Weadapted both Con-1 and HCV-N replicons for use in this system, and haveshown that the induction of SEAP is a useful measure of the replicon RNAabundance in stable, G418-resistant cell lines (FIG. 20), as well as incells that have been transiently transfected by these RNAs (FIG. 21).Parallel measurements of RNA abundance and SEAP expression in twoseparate stable cell lines demonstrated a remarkable degree ofcorrelation (FIG. 20B), providing strong validation of the system.

We have utilized this system to document the inhibition of HCV-N andCon-1HCV RNA replication in En5-3 cells following treatment withrecombinant interferon-α2B (FIG. 22 and FIG. 23). We found Ntat2ANeo(RG)to be about 10-fold less sensitive to interferon than Btat2ANeo(SI).These results differ from those reported recently by Guo et al. (J.Virol., 75, 8516-8523 (2001)), who found comparable interferonsensitivities with simple subgenomic dicistronic replicons constructedfrom these two viral sequences. We are currently investigating themolecular basis of the difference we observed in the interferonresponsiveness of these replicons. Using the tat-expressing replicons,we have also been able to demonstrate the inhibition of viral RNAreplication by prototype antiviral compounds that have activity againstthe viral NS3 proteinase or NS5B RNA-dependent, RNA polymerase. Thus, webelieve that this unique and simple system for monitoring viral RNAreplication is likely to prove useful in future antiviral drug discoveryefforts.

Because measurements of SEAP are technically simpler and considerablyless expensive than quantitative RT-PCR assays for viral RNA, thissystem is likely to prove advantageous for high throughput screening forcompounds with antiviral activity. An additional technical advantageover HCV replicons that express luciferase or most other conventionalreporter proteins is that SEAP activity is measured in supernatantculture fluids and does not require the lysis of cells. This permitsserial measurements of the kinetics of RNA amplification in singlecultures of cells (FIG. 21). One potential drawback of this system isthat suppression of SEAP activity by candidate antiviral compounds couldresult from inhibition of the activity of either the 2A protease or tat,or even (as with other published dicistronic HCV replicons) the EMCVIRES. To address this issue, we established a stably transformed cellline that constitutively expresses the tat2ANeo polyprotein under thetranslational control of the EMCV]RES. This cell line (Et2AN) wasestablished by transfection of pEt2AN DNA (FIG. 16) into En5-3 cells,followed by selection with G418. In contrast to the results shown inFIG. 22, where interferon-α2B suppressed the secretion of SEAP from thereplicon-bearing cell lines, there was no suppression of the secretionof SEAP by the Et2AN cell line at comparable concentrations ofinterferon. This indicates that the effect of interferon-α2B on SEAPsecretion from the replicon cell line was due to specific suppression ofthe replication of HCV RNA, and not the fortuitious suppression of 2A,tat, or EMCV IRES activity. It also demonstrates the absence ofnonspecific toxicity at the concentrations of interferon tested, and isconsistent with the suppression of HCV RNA abundance in these cellsshown in FIG. 23.

In developing these replicons, we have shown that none of the viral coreprotein-coding sequence is required for replication of HCV RNA. Therehas been considerable controversy over the role of this sequence inviral translation since Reynolds et al. (RNA, 2, 867-878 (1996)) firstsuggested that the 5′ proximal 33 nts of the core sequence were anintegral part of the viral IRES and required for efficientcap-independent translation. Recently, however, Rijinbrand et al. (RNA,7, 585-597 (2001)) demonstrated that the requirement is not for anyspecific sequence, but rather for a lack of secondary RNA structurewithin the core-coding sequence immediately downstream of the initiatorAUG. This is consistent with prior work by Honda et al. (RNA, 2, 955-968(1996)) that indicated that stable RNA structure within the vicinity ofthe AUG is very detrimental to IRES-directed translation. Because ofconcerns that the 5′ proximal core coding sequence might be required foroptimal activity of the HCV IRES, the original dicistronic, subgenomicHCV replicons that were constructed by Lohmann et al. (Science, 285,110-113 (1999)) contained RNA encoding 12 or 16 amino acids of the coreprotein fused in-frame to the Neo gene in the upstream cistron. We foundthat replicons in which the tat sequence was fused directly to the HCVIRES had reduced translation of the upstream tat2ANeo mini-polyprotein(FIG. 17A), but were nonetheless capable of replication and thetransduction of G418-resistant cell lines. These results demonstratethat none of the core coding sequence is required for viral RNAreplication. Other subgenomic HCV replicons have recently been describedin which all core protein sequence had been removed, but in thesereplicons translation of the upstream cistron was driven by apicomaviral IRES and the HCV 5′NTR sequence functioned only in templaterecognition by the RNA replicase complex (Kim et al., Biochem BiophysRes Commun, 290, 105-112 (2002)).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the beading, unlessso specified.

1-12. (canceled)
 13. A method for detecting a replication competent HCVRNA, the method comprising: incubating a vertebrate cell comprising anHCV RNA, wherein: the HCV RNA comprises a first coding sequence encodinga hepatitis C virus polyprotein, and a heterologous polynucleotidecomprising a second coding sequence encoding a transactivator; the cellcomprises a transactivated coding region and an operator sequenceoperably linked to the transactivated coding region; and thetransactivated coding region encodes a detectable marker, wherein thetransactivator alters transcription of the transactivated coding region;and detecting the detectable marker, wherein the presence of thedetectable marker indicates the cell comprises a replication competentHCV RNA.
 14. The method of claim 13 wherein the HCV RNA comprises a 3′non-translated RNA, and wherein the heterologous polynucleotide ispresent in the 3′ non-translated RNA or 5′ of the first coding sequence.15. The method of claim 13 wherein the heterologous polynucleotidefurther comprises a third coding sequence encoding a selectable marker,wherein the second coding sequence and the third coding sequencetogether encode a fusion polypeptide.
 16. The method of claim 15 whereinthe heterologous polynucleotide further comprises a fourth codingsequence encoding a cis-active proteinase present between the secondcoding sequence encoding the transactivator and the third codingsequence encoding the selectable marker, wherein the second codingsequence, the fourth coding sequence, and the third coding sequencetogether encode a fusion polypeptide.
 17. canceled
 18. A method fordetecting a replication competent HCV RNA, the method comprising:incubating a vertebrate cell comprising an HCV RNA, wherein: the HCV RNAcomprises a first coding sequence encoding a subgenomic hepatitis Cvirus polyprotein, and a heterologous polynucleotide comprising a secondcoding sequence encoding a transactivator; the cell comprises atransactivated coding region and an operator sequence operably linked tothe transactivated coding region; and the transactivated coding regionencodes a detectable marker, wherein the transactivator alterstranscription of the transactivated coding region; and detecting thedetectable marker, wherein the presence of the detectable markerindicates the cell comprises a replication competent HCV RNA.
 19. Themethod of claim 18 wherein the heterologous polynucleotide furthercomprises a third coding sequence encoding a selectable marker, whereinthe second coding sequence and the third coding sequence together encodea fusion polypeptide.
 20. The method of claim 18 wherein theheterologous polynucleotide further comprises a fourth coding sequenceencoding a cis-active proteinase present between the second codingsequence encoding the transactivator and the third coding sequenceencoding the selectable marker, wherein the second coding sequence, thefourth coding sequence, and the third coding sequence together encode afusion polypeptide.
 21. The method of claim 18 wherein thetransactivator comprises an amino acid sequence comprising at leastabout 70% identity with an amino acid sequence selected from the groupconsisting of SEQ ID NO:19 and amino acids 4-89 of SEQ ID NO:21, andwherein the transactivator has tat activity. 22-45. (canceled)
 46. Amethod for detecting a replication competent HCV RNA, the methodcomprising: incubating a vertebrate cell comprising an HCV RNA, wherein:the HCV RNA comprises a first coding sequence, and a heterologouspolynucleotide comprising a second coding sequence encoding atransactivator; the cell comprises a transactivated coding region and anoperator sequence operably linked to the transactivated coding region;and the transactivated coding region encodes a detectable marker,wherein the transactivator alters transcription of the transactivatedcoding region; and detecting the detectable marker, wherein the presenceof the detectable marker indicates the cell comprises a replicationcompetent HCV RNA.
 47. The method of claim 46 wherein the first codingsequence encodes a subgenomic hepatitis C virus polyprotein.
 48. Themethod of claim 46 wherein the first coding sequence encodes a hepatitisC virus polyprotein.